Extended use planar sensors

ABSTRACT

A planar, solid-state electrochemical oxygen sensor having a substrate, conductive strips deposited on the substrate, and a dielectric layer insulating portions of the conductive strips except those portions which define a working electrode and at least one second electrode. The working electrode may be defined by an open printed region of the dielectric, or by a needle-punched or laser-burned hole or opening in the dielectric which exposes a small region of one of the conductive strips. A solid electrolyte contacting the electrodes is covered by a semipermeable membrane which may comprise an acrylonitrile butadiene copolymer or an acrylate-based copolymer. A sample chamber is defined by the membrane, a cover member, and a gasket therebetween, and has a volume of from about 1 to about 2 μl. The gasket is formulated from the highly cross-linked polymerization product of epichlorohydrin. All sensor components are selected such that a sensor operable for at least 2 days under normal conditions is produced.

This is a divisional of patent application Ser. No. 08/045,847 filed onApr. 9, 1993, now U.S. Pat. No. 5,387,329.

FIELD OF THE INVENTION

The present invention relates generally to planar sensors, and morespecifically to a planar electrochemical oxygen sensor that is small, isnon-oxygen-depleting, is convenient and inexpensive to manufacture, andhas a fast response time and has a long useful life.

TECHNICAL REVIEW

In the clinical setting, it is important to monitor certain bloodanalytes which are significant to normal physiological function andhomeostasis. Such blood analytes include pCO₂, pO₂, tHb, pH, Na⁺, K⁺,Cl⁻ and Ca²⁺, The focus of the present invention is to sensors for themeasurement of pO₂, i.e., the partial pressure of oxygen. The teachingof the present invention further extends to sensors for measuring otherblood analytes and to sensors for use in other technical fields.Conventional approaches for doing so utilize a variety ofelectrochemical means such as two or three-electrode electrochemicalsensors having liquid, solid, or gel electrolytes.

Conventional sensors are fabricated to be large, comprising manyserviceable parts, or to be small, planar-type sensors which may be moreconvenient in many circumstances. The term "planar" as used hereinrefers to the well-known procedure of fabricating a substantially planarstructure comprising layers of relatively thin materials, for exampleusing the well known thick-film or thin-film techniques. See, forexample, U.S. Pat. No. 4,571,292, and U.S. Pat. No. 4,536,274, bothincorporated herein by reference.

The electrodes of such a sensor are conventionally addressed by anelectrolyte covered by a gas permeable but liquid impermeable membranesuch as teflon, polyethylene or polypropylene. As blood is placed incontact with the membrane, oxygen from the blood diffuses across themembrane, through the electrolyte solution, and is reduced at a cathode.The oxygen partial pressure of the sample is determined by measuring theresultant electrical current flowing through a circuit including thecathode and an anode. A three-electrode sensor may be employed, whichmaintains a constant voltage relationship between a working electrode(cathode) and a reference electrode by a feedback control, and isdescribed in U.S. Pat. No. 4,571,292, referenced above. According tosuch a system, oxygen may be reduced at the working cathode, thisreaction driven by the constant potential between the working andreference electrodes such that a reaction taking place at a counterelectrode (anode) is simply the reverse of that occurring at the workingcathode, that is, a non-electrode-consuming reaction.

With regard to both two and three-electrode sensors, it is desirable tomanufacture a working electrode (cathode) to be very small. In this way,reduction of oxygen at the cathode is the rate-limiting step, and moreaccurate current output as a function of pO₂ is realized.

Additionally, the cathode is desirably manufactured to be very small sothat the sensor exhibits non-depleting behavior, that is, fast, stirrate independent (time-independent) current response is realized. Theprincipal of non-depletion is discussed in an article entitled"Voltammetric Microelectrodes", in Current Separations, 8, 1/2 (1987) byJonathan O. Howell, and references therein. However, it is difficult toroutinely and inexpensively manufacture a sensor having a workingelectrode small enough so as to be non-depleting.

In the clinical setting it is a goal, with respect to electrochemicalblood analyte analysis, to maximize the data obtainable from a samplehaving a volume on the order of microliters. Fabrication of a very smallsensor sample chamber for holding a sample in contact with asemipermeable membrane is desirable in this regard so that many testsmay be performed, for example using a series of interconnected sensorseach constructed to detect a different analyte, from one very smallsample volume such as a capillary tube sample. However, as a samplechamber is made smaller, the concentration of contaminations in a samplefrom sensor components themselves, especially components defining asample chamber, and/or certain reaction products of sensor functionitself, is increased. Such contamination may result in premature sensorfailure.

The lifetime of small planar sensors is commonly dictated bycontamination of a semipermeable membrane by sample components or byimpurities present in other sensor components which may leach into themembrane directly or via the sample. Such contamination commonly affectsthe permeability characteristics of the membrane, which may affect thelinearity of sensor performance, or may allow the passage of specieswhich may contaminate underlying electrolyte or electrodes.

An additional factor which may shorten sensor lifetime is delaminationof various sensor components from other sensor components. Thesecomplications typically result in sensors that do not provide preciseand reproducible current output at a given level of analyteconcentration to which the sensor is exposed for an acceptable period oftime. Thus, commercially-available planar electrochemical sensors formeasuring analytes in blood such as oxygen generally fail after exposureto a limited number of samples.

Indicators which has been found by the inventors to determine sensorutility, and to monitor the useful life of a sensor, include sensorpolarograms which differ drastically from those which the sensorexhibited when new and/or which contain spurious peaks attributable toimpurities, and plots of current output versus analyte concentrationwhich show unacceptable drift.

If it were possible to fabricate a semipermeable membrane for separatinga sample area from an electrochemical sensing area that was relativelyimpermeable to species detrimental to the sensor, that was unaffected byany other sensor components, and was effective for a long period oftime, sensor longevity would be increased.

Attempts have been made, for example, U.S. Pat. No. 4,734,184, issuedMar. 29, 1988, incorporated herein by reference, describes a disposableelectrochemical sensor apparatus for measuring analytes in body fluidsamples. A membrane is constructed to separate the sample area from theelectrochemical sensor area and to pass analytes and species necessaryfor the sensing function, while preventing transport of undesirablespecies.

However, it remains a challenge in the art to formulate a membranecomposition which exhibits a constant permeability to desired speciesover an extended period of time while maintaining impermeability toundesired species for the same length of time. Additionally, it is anadded challenge to engineer into such a membrane desirable macroscopicphysical properties such as durability and flexibility, whilemaintaining desirable microscopic physical properties that providedesirable permeability characteristics of the membrane.

Accordingly, it is a general purpose of the present invention to providea means and method of measuring the concentration of anelectrochemically active species such as oxygen in a fluid such asblood, using a small electrochemical sensor which is relatively simpleand inexpensive to manufacture, is non-depleting with respect to theactive species measured, and has a relatively long useful life.

SUMMARY OF THE INVENTION

The foregoing and other objects and advantages of the present inventionare achieved by providing a semipermeable membrane for use in a planaroxygen sensor or the like, comprising a polymer which may be depositedfrom organic solution and which itself comprises the polymerizationproduct of at least one nitrile monomer unit and at least one conjugateddiene monomer unit. In an alternate embodiment, the membrane comprisesthe polymerization product of at least one acrylate monomer unit. Themembrane is desirably impermeable to liquid water, but rapidly permeableto water vapor. Additionally, it is preferred that the membrane for anoxygen sensor be impermeable to ions and other blood constituents, andhave limited permeability to oxygen. The membrane functions usefully fora period of at least 2 days, preferably 10 days, and more preferably 15days under normal sensor operation when used with the inventive oxygensensor. Sensors fabricated in accordance with a particularly preferredembodiment, containing a particularly preferred membrane, have beendemonstrated to function usefully for at least 60 days under normaloperation. Normal sensor operation is defined hereinbelow.

It is another object of the present invention to provide a gasket forforming a liquid and gas impermeable seal between a semipermeablemembrane and a cover member which together define in part a samplechamber of an electrochemical sensor. The gasket comprises a highlycross-linked polymerization product of an epoxy compound and ahydrophilic monomer according to one embodiment. Preferably, the gaskethas a Shore A hardness of from about 10 to about 100, an oxygenpermeability of less than 20 Barrers, and is liquid impermeable andformulated to be substantially free of mobile extractable materialswhich could leach into the membrane affecting physical or permeabilitycharacteristics therein. The gasket is further formulated to besubstantially free of mobile sulfides and battery metals, and iseffective in forming and maintaining said seal for at least 2 days,preferably 10 days, and more preferably 15 days under normal sensoroperation.

It is another object of the present invention to provide a samplechamber for containing a physiological fluid, the PO₂ of which is to bemeasured, which chamber is defined by a semipermeable membraneoverlaying an electrochemical sensor, a cover member having at least onepassageway through which the sample fluid may pass, and at least onegasket between the cover member and the membrane which seals thechamber. The gasket is selected so as to be free of any extractablematerials, or such that any extractable materials present do not leachinto the membrane in a way that affects the desirable features of themembrane. The gasket is further selected so as to be substantially freeof species which, if they were to leach into a sample, would interferewith the electrochemical measurement of a particular analyte therein, orwhich would adversely affect the electrolyte, electrodes, or othercomponents of the electrochemical sensor. According to this embodiment,the gasket and membrane are further selected so as to have sufficientflexibility characteristics to form a sealed junction at theirinterface, which junction remains sealed for the lifetime of the sensor,and which seal does not adversely physically affect the gasket or themembrane.

It is another object of the invention to provide a novel means andmethod for forming a small, electrically addressable area ofelectrically conductive material for use as a microelectrode or thelike. According to the method, an electrically conductive material isplaced on a substrate, and at least a portion of the material coveredwith an electrically-insulating non-porous material, i.e., a dielectricmaterial. A small discontinuity is formed in the electrically insulatingmaterial using a needle punch to expose a small,electrically-addressable area of the electrically conductive material,and defining a microelectrode. According to a specific method, a needlepunch is in electric communication with the electrically conductivematerial. When the needle punch penetrates the electrically insulatingmaterial and contacts the electrically conductive material, anelectrical circuit is formed, indicating such penetration, and theneedle punch is withdrawn from the electrically conductive material. Theapparatus may be designed so that the needle is withdrawn at any of avariety of positions relative to the surface of the electricallyconductive material.

It is another object of the present invention to provide a means andmethod for forming a two or three-electrode solid-state planar oxygensensor using thick or thin-film techniques, which sensor isnon-depleting, is relatively inexpensive, has a fast response time, isconstructed to measure blood samples having volumes on the order ofmicroliters, and has a lifetime on the order of days or months.

It is another object of the present invention to provide a solid-stateplanar oxygen sensor comprising an electrically nonconductive substrate,a first electrically conductive material adhered to a first portion ofsaid substrate, at least a portion of said first conductive materialbeing covered with an electrically insulating dielectric coating and atleast one portion of said first conductive material remaining uncoveredby said electrically insulating dielectric coating, said uncoveredportion of said first conductive material defining a working electrodearea, a second electrically conductive material adhered to a secondportion of the substrate, a portion of said second conductive materialbeing covered with the electrically insulating dielectric coating and atleast one portion of said second conductive material remaining uncoveredby said electrically insulating dielectric coating, said uncoveredportion of said second conductive material defining a counter electrodearea, a third electrically conductive material adhered to a thirdportion of the substrate, a portion of said third conductive materialbeing covered with the electrically insulating dielectric coating and atleast one portion of said third conductive material remaining uncoveredby said electrically insulating dielectric coating, said uncoveredportion of said third conductive material defining a reference electrodearea, a hygroscopic solid electrolyte provided on at least the workingelectrode area and the counter electrode area, said electrolyte having aswell value of less than two times its dry volume when provided withwater, a semipermeable membrane covering said electrolyte having anoxygen permeability of less than 8 Barrers, a cover member covering saidsemipermeable membrane and having a recess formed therein, said recesshaving a perimeter facing the membrane and at least one passagewayconnected to the recess, and a gasket contacting the recess perimeterand the membrane to form a seal therebetween, the gasket, cover member,and membrane defining a sample chamber.

It is another object of the present invention to provide a solid-stateplanar oxygen sensor comprising an electrically nonconductive substrate,a first electrically conductive material adhered to a first portion ofsaid substrate, at least a portion of said first conductive materialbeing covered with an electrically insulating dielectric coating and atleast one portion of said first conductive material remaining uncoveredby said electrically insulating dielectric coating, said uncoveredportion of said first conductive material defining a working electrodearea, a second electrically conductive material adhered to at least asecond portion of the substrate, a portion of said second conductivematerial being covered with the electrically insulating dielectriccoating and at least one portion of said second conductive materialremaining uncovered by said electrically insulating dielectric coating,said uncovered portion of said second conductive material defining acounter electrode area, an electrolyte provided on at least the workingelectrode area and the counter electrode area, and polymericsemipermeable membrane covering the electrolyte, said membranecomprising the polymerization product of at least one monomer having theformula CH₂ ═C(R₁)(COOR₂), where R₁ and R₂ are each selected from thegroup consisting of hydrogen, hydrocarbon groups, and alcohol groups andR₁ and R₂ can be the same or different, or the polymerization product ofat least one nitrile-containing monomer and at least one conjugateddiene monomer.

It is another object of the present invention to provide anelectrochemical sensor comprising electrochemical means for detecting ananalyte in a test sample, a semipermeable membrane covering saidelectrochemical means, said membrane being permeable to said analyte andto species necessary for the functioning of said electrochemical means,a cover member covering said semipermeable membrane and having a recessformed therein, said recess having a perimeter facing the membrane andat least one passageway connected to the recess, and a gasket contactingthe recess perimeter and the membrane to form a seal therebetween, thegasket, cover member, and membrane defining a sample chamber.

It is another object of the present invention to provide a method forforming a microelectrode for use as a working electrode in a planar,solid-state oxygen sensor or the like, comprising the steps of selectingan electrically nonconductive substrate, combining said substrate withan electrically conductive material with said electrically conductivematerial forming a layer adjacent said electrically nonconductivesubstrate, coating at least a portion of the electrically conductivematerial with an electrically insulating material, puncturing theelectrically insulating material to form at least one hole or opening oropening therein with a needle in communication with electric circuitmeans in communication with the electrically conductive material,moveable between a first position and a second position in which theelectric circuit means generates a signal withdrawing the needle whenthe signal is generated, said insulating material being selected so asto firmly adhere to the conductive material such that when the hole(s)or opening(s) is formed an exposed, electrically addressable portion ofthe electrically conductive material is created, defining at least oneelectrode having a size defined by the cross-sectional area of the holeor opening.

It is another object of the present invention to provide a method forforming a microelectrode for use as a working electrode in a planar,solid-state oxygen sensor or the like, comprising the steps of selectingan electrically nonconductive substrate, combining said substrate withan electrically conductive material with said electrically conductivematerial forming a layer adjacent said electrically nonconductivesubstrate, coating a surface of the electrically conductive materialwith an electrically insulating material selected so as to firmly adhereto the conductive material, passing a needle in communication withelectric circuit means in communication with the electrically conductivematerial through the electrically insulating material, which needle ismovable between a plurality of positions relative to the surface, someof which positions cause the electric circuit means to generate signals,and withdrawing the needle when a predetermined signal is generatedindicating a predetermined relative position.

It is another object of the present invention to provide anelectrochemical oxygen sensor having an improved semipermeable membranecomprising the polymerization product of at least one nitrile-containingmonomer and at least one conjugated diene monomer.

It is another object of the present invention to provide anelectrochemical sensor having an improved semipermeable membranecomprising the polymerization product of at least one monomer having theformula CH₂ ═C(R₁)(COOR₂), where R₁ and R₂ are each selected from thegroup consisting of hydrogen, hydrocarbon groups, and alcohol groups andR₁ and R₂ can be the same or different.

It is another object of the present invention to provide a method forselecting an electrically-conductive material for use in anelectrochemical sensor or the like, comprising measuring the polarogramof said material to determine the shape of said polarogram in apredetermined potential region within which said sensor is designed tooperate, and evaluating said polarogram in said predetermined region todetermine whether said polarogram is substantially free of excessivepeaks.

It is another object of the present invention to provide anelectrochemical sensor having a semipermeable membrane, electrochemicalmeans on a first side of said membrane for detecting an analyte in asample, and a sample chamber on a second side of said membrane forreceiving said sample, said chamber defined in part by said second sideof said membrane and a cover member having a recess formed therein, saidrecess having a perimeter facing said second side of said membrane, andbeing improved by having a gasket for forming a liquid and gasimpermeable seal between said second side of said semipermeable membraneand said cover member recess perimeter, said gasket comprising a durableorganic polymer or copolymer which does not creep or flow when stressed,which has a low durometer rating, which is gas impermeable, and which isslightly hygroscopic and thus swells slightly in the presence ofsolution containing water.

It is another object of the present invention to provide a compositioncomprising the polymerization product of at least one monomer having theformula CH₂ ═C(R₁)(COOR₂), where R₁ and R₂ are each selected from thegroup consisting of hydrogen, hydrocarbon groups, and alcohol groups andR₁ and R₂ can be the same or different, at least one monomer selectedfrom the group consisting of those having the formulas CH₂ ═C(R₁)(CONR₂R₃), CH₂ ═C(R₁)(OCOR₂), where R₁, R₂, and R₃ are each selected from thegroup consisting of hydrogen, hydrocarbon groups, and alcohol groups andR₁, R₂, and R₃ can be the same or different, and at least onenitrile-containing monomer.

It is another object of the present invention to provide a compositioncomprising the polymerization product of at least one monomer having theformula CH₂ ═C(R₁)(COOR₂), where R₁ is selected from the groupconsisting of hydrogen and lower alkyl groups, R₂ is selected from thegroup consisting of linear, branched and cyclic hydrocarbons andalcohols of from 1 to 20 carbon atoms, R₁ and R₂ being the same ordifferent, said monomer being present in an amount of from about 20% toabout 80% by weight based on the weight of the composition at least onenitrile-containing monomer, said nitrile-containing monomer beingpresent in an amount of from about 15% to about 80% by weight, and atleast one monomer selected from the group consisting of those having theformula CH₂ ═C(R₁)(OCOR₂) where R₁ and R₂ are each selected from thegroup consisting of hydrogen, lower alkyl groups and lower alcohols andR₁ and R₂ can be the same or different, those having the formula CH₂═C(R₁)(CONR₂ R₃) where R₁, R₂, and R₃ are each selected from the groupconsisting of hydrogen and lower alkyl groups and R₁, R₂, and R₃ can bethe same or different, and a combination of the two.

It is another object of the invention to provide a method for evaluatingthe adherence between an electrically conductive material and anelectrically insulating dielectric layer contacting the electricallyconductive material, the dielectric material arranged so as to expose aspecific region of said electrically conductive material defining anelectrode, comprising biasing the electrode at a potential with a secondelectrode, said electrode and second electrode being contacted by anelectrolyte; holding the potential constant over a predetermined periodof time; measuring the resultant current output at said potential oversaid period of time; and determining whether said current output issubstantially steady and substantially free of variation.

It is another object of the invention to provide a method of adjustingthe oxygen permeability of a semipermeable membrane comprising acopolymer of a nitrile-containing monomer and a conjugated diene,comprising: adding a predetermined quantity of a species selected fromthe group consisting of a vinyl halide, a vinylidene halide, a copolymercomprising vinyl halide and a nitrile-containing monomer, a copolymercomprising vinylidene halide and a nitrile containing monomer, or acombination of any of the above, to said nitrile/conjugated dienecopolymer, to form a mixture of copolymers, said predetermined amountbeing selected so as to adjust the oxygen permeability to apredetermined level.

It is another object of the invention to provide a method for evaluatingthe durability of an electrochemical sensor comprising: measuring thepolarogram of said sensor to determine the shape of said polarogram in apredetermined potential region within which said sensor is designed tooperate, and evaluating said polarogram in said predetermined region todetermine whether said polarogram is substantially free of excessivepeaks.

These and other objects in view, as will be apparent to those skilled inthe art, the invention resides in the combination of elements set forthin the specification and covered by the claims appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features, objects and advantages of the presentinvention will be better understood from the following specificationwhen read in conjunction with the accompanying drawings, in which:

FIG. 1 is a top view of a partial assembly of a preferred embodiment ofa planar oxygen sensor according to the present invention withelectrolyte, membrane, gasket and cover member removed;

FIG. 2 is a top view of a partial assembly of an alternate embodiment ofa planar oxygen sensor according to the present invention withelectrolyte, membrane, gasket and cover member removed;

FIG. 3 is a cross-sectional view through line 3--3 of FIG. 1, withelectrolyte, membrane, gasket and cover member in place;

FIG. 4 is a cross-sectional view through line 4--4 of FIG. 2, withelectrolyte, membrane, gasket and cover member in place;

FIG. 5 is a cross-sectional view through line 5--5 of FIGS. 3 or 4showing this view of the embodiments of FIGS. 3 and 4 to be identical;

FIG. 6 is a graph of membrane permeability as a function of percentpolyvinyl chloride or poly(vinylidene chloride-co-acrylonitrile),according to selected embodiments of the present invention;

FIG. 7 is a graph of current vs. time illustrating the current responseto PO₂ in aqueous calibrators, measured according to one embodiment ofthe invention;

FIG. 8 is a graph of current vs. time illustrating the current responseto PO₂ in blood samples, measured according to one embodiment of thepresent invention;

FIG. 9 illustrates linear sweep voltamograms, after 16 days use, ofsensors fabricated according to three embodiments of the presentinvention;

FIG. 10 illustrates sensor response to aqueous oxygen calibratorsolutions, after 16 days use, of sensors fabricated according to threeembodiments of the present invention;

FIG. 11 illustrates linear sweep voltamograms, after 60 days use, ofsensors fabricated according to three embodiments of the presentinvention;

FIGS. 12A-12G illustrate cyclic voltamograms, taken after variousperiods of use, of a sensor fabricated according to one embodiment ofthe present invention;

FIGS. 13A-13G illustrate cyclic voltamograms, taken after variousperiods of use, of a sensor fabricated according to one embodiment ofthe present invention;

FIGS. 14A-14H illustrate cyclic voltamograms, taken after variousperiods of use, of a sensor fabricated according to one embodiment ofthe present invention;

FIG. 15 illustrates sensor response to aqueous oxygen calibratorsolutions and to tonometered whole human blood samples according to oneembodiment of the present invention; and

FIG. 16 illustrates polarograms, taken after five days of sensor testingusing oxygen calibrator solutions, human serum, and tonometered humanwhole blood samples, according to one embodiment of the presentinvention.

DESCRIPTION OF PREFERRED EMBODIMENTS

A planar oxygen sensor 30 in accordance with a preferred embodiment ofthe present invention is illustrated in FIG. 1 and includessubstantially planar substrate 32, conductive metal strips 34, 36, and38 deposited thereupon, and dielectric layer 40 deposited on substrate32 so as to cover portions of conductive strips 34, 36, and 38, whileleaving portions of some of the conductive strips uncovered.

Substrate 32 is formed from any substantially electrically insulatingmaterial such as ceramic, glass, refractory, polymers or combinationsthereof. Formation of such an insulating substrate as a mechanicalsupport or base is common knowledge to those of ordinary skill in theart. In the preferred embodiment, the substrate comprises approximately96% alumina and approximately 4% glass binder. A suitable materialcomprising the preferred composition is available from Coors CeramicCompany, Grand Junction, Colo. Although in the preferred embodiments ofthe present invention a single substrate forms the foundation of theoxygen sensor, a plurality of substrates each supporting separate sensorcomponents, and/or helping to support sensor components supported byother substrates, could be employed.

Conductive strips 34, 36, and 38 are deposited atop substrate 32 so asto extend from a first end 41 thereof to a second end 43 thereof in apreferred embodiment. At first end 41, the conductive strips aretypically deposited so as to be wide enough to define contact pads 42,44, and 46, respectively. At second end 43, the conductive strips aretypically deposited so as to be somewhat narrower, exposed regions ofwhich may define electrodes, as described below.

Conductive strips 34, 36, and 38 may be deposited using the well knownthin or thick-film techniques. Typically, a compound including a metalis applied via typical thick-film screening to substrate 32, and theapplied compound and substrate are then fired to sinter the active metaland to co-adhere the active metal to the substrate. The electroactivemetal may comprise any conductive metal, for example, silver, platinumor gold, which is not oxidized or reduced in a potential range in whichoxidation or reduction of any species to be measured occurs. In the caseof an oxygen sensor, electroactive metals should not be susceptible tooxidation in the range of from 0 to -1200 millivolts (mV) vs.silver/silver chloride. Additionally, materials selected for fabricationof conductive strips 34, 36, and 38 are desirably selected so as to befree of any impurities such as battery metals (that is, ones that areelectrochemically active in water) which are typically present inoff-the-shelf materials commercially available for wire bonding, etc.

Many thick-film pastes are commercially available, such as a silverpastes available as part number 3571UF/Ag from Metech Company, ofElverson, Pa. (Metech), and part number 6061/Ag from E.I. DuPont DeNemours & Company (DuPont) of Wilmington, Del.; silver chlorideavailable as part numbers 2539/Ag/AgCl or PC10299 from Metech; goldpastes available as part number PC10231/Au from Metech, part number5715H/Au from DuPont, and part number JM1301/Au from Johnson Matthey ofWest Deptford, N.J.; and platinum paste available as part numberPC10208/Pt from Metech.

In the selection of specific pastes for use as electrodes in theinvention, and in one embodiment electrodes may be continuous withconductive strips, and with specific regard to conductive strip 38 whichdefines in part a working electrode, as described below according to oneembodiment, it is advantageous to conduct a preliminary evaluation inwhich a polarogram exhibited by the working electrode material isrecorded in order to assess manufacturing, quality control, and productspecifications. As is well known, a polarogram may be recorded bybiasing an electrode against a reference in the presence of anelectrolyte, varying the voltage within a predetermined range, andrecording the current output as a function of voltage.

As discussed above, it has been determined by the inventors that adetermination of sensor utility may be made based on indicatorsincluding polarograms exhibited by the sensor. Additionally, it has beendetermined in accordance with the present invention that a determinationof a suitable candidate for use as a material for fabrication of theelectrodes, especially a working electrode (described below) and in oneembodiment conductive strip 38, may also be made based upon a polarogramexhibited by the material. A suitable candidate as a material forfabrication of an electrode, especially a working electrode (andconductive strip 38 according to one embodiment) advantageously willexhibit a polarogram having a plateau in a region in which the sensorwill typically be polarized during electrochemical analysis. Accordingto a preferred embodiment of the present invention, this means that asuitable material will exhibit a relatively flat current vs. voltageplot in the region of approximately -0.800 V±0.300 volts (V) vs.silver/silver chloride. It is to be understood that the choice ofelectrical potential in electrochemical analyses of sensor components,for example polarogram-based analyses, is not critical to the presentinvention. Any potential setting may be chosen in accordance with thedesirable operational parameters described herein.

If, during sensor operation, the voltage at which the working electrodewas set could be held absolutely constant, there would be no need for anelectrode material polarogram plateau. However, as absolute voltagecontrol is not practical, a plateau is desirable. As used herein, theterms "plateau" and "relatively flat current vs. voltage plot" are meantto define a region of the curve defined by the polarogram in whichinherent instrumental and experimental voltage fluctuation does notresult in current readout which fluctuates outside of an acceptablemargin of error, in the operation of some sensors a plateau may not beobserved but function properly as long as voltage fluctuation does notvary outside an acceptable margin of error.

It has also been determined in accordance with the invention thatevaluation of material for electrode use based on the above-notedpolarogram analysis is advantageous as a polarogram may exhibit peaksindicative of impurities present in the material. It is to be understoodthat a peak in such a polarogram is not necessarily indicative of anunsuitable material. For example, a peak may merely indicateelectrochemistry associated with the consumption of an impurity, andindicating a material well-suited for electrode use. However, a peak maybe indicative of the presence of an impurity generated from, or within,the material during the electrochemical measurement, and thus mayindicate that a material is unsuitable.

With specific regard to conductive strip 38, which defines in part aworking electrode, a preferred material is a very high purity platinumthick-film paste. Conductive strip 34 preferably comprises a thin layerof silver deposited atop substrate 32 with a layer of silver chloridedeposited thereupon in the electrode region, discussed below, to createa reference electrode. Alternately, a silver layer deposited asconductive strip 34 may be electrochemically oxidized in the presence ofsufficient chloride ion to form a silver chloride layer atop a silverlayer for use as a reference electrode. Such deposition orelectrochemical formation of a silver/silver chloride referenceelectrode is well known in the art. Conductive strip 36 comprises a goldthick-film paste or a platinum thick-film paste in preferredembodiments.

Employment of a silver reference electrode is within the scope of thepresent invention. Modification of the teachings of the presentinvention with respect to voltage settings, upon the substitution of asilver reference electrode for a silver/silver chloride referenceelectrode, would be easily made by one of ordinary skill in the art.

At the second end 43 of substrate 32, dielectric layer 40 is depositedso as to cover portions of conductive strips 34, 36, and 38, whilecontaining an open printed region 48 which leaves portions of theconductive strips uncovered so as to define reference electrode 50, (ifpresent), counter electrode 52, and working electrode 54. Materialselected for fabrication of the dielectric layer 40 is desirablyelectrically insulating and non-porous, free of impurities which may besubject to oxidation or reduction in the potential range of any speciesor analyte to be measured, as described above, and is further selectedso as to be free of mobile ions that would potentially carry charge andinterfere with the activity of any electrolyte employed in the sensor.Further, dielectric 40 is selected so as to firmly adhere to substrate32 and conductive strips 34, 36, and 38, so as to allow electrodes 50,52, and 54 to be electrically addressable, while effectivelyelectrically insulating portions covered by the dielectric. Materialssuch as ceramics, glass, refractory materials, polymeric materials, orcombinations thereof are well known as dielectric materials and aresuitable for use as a dielectric in the present invention.

With respect to materials advantageously selected for fabrication ofconductive strips 34, 36, and 38, it is noted that material selectionbecomes less important in regions of the strips which define contactpads 42, 44, and 46 and which connect the bonding pads to regions whichdefine electrodes. For example, the contact pads and regions of theconductive strips connecting them to the electrodes may be fabricatedfrom any conducting material that adheres to substrate 32 and that doesnot interfere with the electrical insulation function of dielectriclayer 40. According to one embodiment, the contact pads and regions ofthe conductive strips connecting them to the electrodes are fabricatedfrom a gold paste.

In addition to the material selection parameters discussed above, and asdiscussed with respect to selection of the dielectric material, it isadvantageous in the fabrication of an extended-use sensor to selectmaterials for fabrication of the substrate, the conductive strips, andthe dielectric layer such that good adherence is achieved betweenadjacent layers, that is, delamination is minimized. If good adherenceis not achieved reference, counter, and working electrodes 50, 52, and54 will not be well-defined by open printed region 48, in one embodimentas defined by a screen used in the thick-film deposition process, anddisadvantageous electrochemistry will result.

Preliminary electrochemical analysis may also be used to evaluateadherence between various materials according to the following method.An electrolyte is applied to the reference, counter, and workingelectrodes 50, 52, and 54 of the sensor as described above. The sensoris biased at a potential between the working electrode and the counterelectrode, and a resultant current output level at constant voltage isobserved over time. If the current output level is substantially steadyand substantially free of variation, good adherence between adjacentlayers is indicated. If delamination occurs, specifically, delaminationbetween dielectric layer 40 and conductive strips 34, 36, and/or 38where open printed region 48 defines electrodes 50, 52, and 54, this maybe indicated by spurious peaks and other discontinuities in the currentoutput level. Typically, such peaks and/or discontinuities take the formof a sharp, spontaneous rise from a baseline steady-state current,followed by gradual current tapering to a slightly higher steady-statecurrent than the baseline current. Thus, testing of sensors andelectrodes in such a manner gives rapid indication as to whether or nota particular combination of insulating material and conductive materialshows sufficient adherence to define an electrode, particularly a small,working electrode in accordance with the methods of the presentinvention. Generally, selection of materials for deposition as adjacentlayers which have similar coefficients of linear expansion may preventdelamination to some extent.

An alternate embodiment of the present invention is illustrated at 130in FIG. 2. In FIG. 2 and in all the accompanying figures, elements ofthe present invention common to several figures are represented bycommon numerical designations. Referring to FIG. 2, dielectric layer 40is deposited so as to expose portions of conductive strips 34 and 36,defining reference electrode 50 and counter electrode 52, respectively,but conductive strip 38 is entirely covered by dielectric 40 at thesecond end 43 of substrate 32. At least one small hole(s) or opening(s)56 is then formed in the dielectric layer 40 so as to expose an area ofconductive strip 38 which defines a working electrode 154.

Hole(s) or opening(s) 56 may be formed according to a variety ofmethods. According to one method, a laser is fired at dielectric layer40 to form hole or opening 56. The laser and dielectric may be selectedsuch that the wavelength of radiation emitted by the laser is absorbedby dielectric 40 to a degree sufficient to efficiently form the hole oropening 56 therein. A dielectric selected so as to absorb laserradiation to the greatest extent possible will result in a hole oropening 56 having a shape that corresponds closely with the shape of thecross-sectional area of the laser beam, as the hole or opening may beburned rapidly and efficiently. Formulating a dielectric such that thewavelength of radiation emitted by the laser is absorbed to a highdegree may be effected by, for example, introducing pigments into thedielectric formulation. Such introduction of pigment should be made inaccordance with the teachings herein regarding mobile species which mayhave an electrochemical effect or a physical effect on sensorcomponents. A clear dielectric may also be selected, in which case holeor opening 56 would be formed as laser radiation would be absorbed byconductive strip 38, and overlaying dielectric layer 40 would be heatedthereby. Thus, according to the method, a cross-sectional laser beamarea being known, a working electrode having a predetermined size andshape may be defined.

Although hole or opening 56 is generally substantially circular whenformed according to this embodiment, it need not be. For example, alaser may be oriented such that the laser beam strikes dielectric layer40 at an angle other than a perpendicular angle. In such a case, hole oropening 56 may be elongated or oval-shaped. Additionally, the laser neednot be stationary during firing, but may be moved during firing as toform an elongated or line-shaped hole. A plurality of holes or openingsmay be formed as well to create a plurality of small working electrodes.Such manipulation in concert with a particular selection of wavelengthand laser focal adjustment may advantageously be utilized to create anydesired size and shape of hole or opening 56 defining working electrode154.

According to another method of formation of hole(s) or opening(s) 56,and referring still to FIG. 2, a needle is used to puncture dielectric40 above conductive strip 38. As used herein, the term "needle" isunderstood to mean any instrument such as a needle, drill bit,ultrasonic tool, or the like. According to a preferred embodiment, atungsten carbide needle having a tip radius of about 12 microns isemployed. As noted herein, it is desirable to form a very small hole oropening 56 such that a very small working electrode 154 may be defined.However, using a needle to puncture material to expose an underlyinglayer may result in deformation of the surface exposed. Specifically,unless the needle is immediately withdrawn as soon as it contacts thesurface of the material to be exposed, a depression in the material maybe formed by the tip of the needle, resulting in essentially greatersurface area exposed. Such precise needle control, especially if theneedle is to puncture a series of holes or openings in impreciselypositioned articles, may be a complication.

Therefore, according to one method, a needle is provided incommunication with electric circuitry, which electric circuitry is alsoin contact with conductive strip 38 via contact pad 46. Dielectric 40 ispunctured by the needle until the needle contacts conductive strip 38,closing an electrical circuit including the needle, conductive strip 38,and the electric circuitry. When the circuit is closed, a signal is sentto controlling apparatus which immediately withdraws the needle fromdielectric 40. In this way, the size of hole or opening 56, and theresultant size of electrode 154, is minimized and controlled. Theelectrical circuitry employed may be any conventional analog or digitalcircuitry, and is advantageously controlled by computer.

According to a preferred method, sensor 130 is mounted on an X-Y-Z tablesuch as are available from Asymtek Corporation of Carlsbad, Calif., andis positioned such that a needle may puncture dielectric 40 directlyabove conductive strip 38. According to one embodiment, a needleassembly is mounted on a weighted ball slide which may be adjusted withrespect to the downward force applied onto the needle by removing oradding weight. The needle and conductive strip 38 are connected byelectric circuitry in communication with a computer, and when the needlecontacts conductive strip 38, the computer commands the ball slide motorto immediately reverse direction and the needle is immediatelywithdrawn. It is to be understood that other methods of controlleddownward force on the needle are within the scope of the presentinvention. For example, the needle may be mounted on apiezoelectrically-controlled device, such devices typically beingcapable of controlled motion to within one mm. Other modifications ofdownward force control known to those of ordinary skill in the art couldbe employed as well.

The method is applicable to the formation of microelectrodes of avariety of types, designed for a variety of uses, including other usesin the microelectronic arts, for example, any electrically conductivematerial may be coated with an electrically insulating material, and ahole or opening punctured in the electrically insulating material toform the electrode. The needle may be movable between a first positionin which the needle is not in contact with the electrically insulatingmaterial, and a second position in which the needle has punctured theelectrically insulating material and is contact with the electricallyconductive material, at which point an electric circuit is closed,generating a signal which actuates apparatus causing the needle to bewithdrawn.

Alternatively, the needle may be movable between a plurality ofpositions relative to the surface of the electrically conductivematerial, some of which positions cause an electric circuit in contactwith the needle and the conductive material to generate signals. Forexample, signals may be generated only at particular thresholdresistivity levels, and in this manner, the depth of penetration of theneedle into the conductive material may be regulated by allowing asignal generated at the desired depth of penetration to actuateapparatus causing the needle to be withdrawn. In this manner, selectinga conductive material which may be penetrated to some extent by theparticular needle selected may result in needle penetration, and aresultant depression may be formed in the surface of the conductivematerial to one of a variety of predeterminable depths, defining avariety of predeterminable electrode areas. In this manner,microelectrodes having precisely controlled surface areas may beproduced. Control of the depth of needle penetration into theelectrically conductive material may be based upon resistivity of acircuit including the needle and the electrically conductive material,as mentioned above, or may be based upon adjusting the ratio of theweight applied to a weighted ball slide to the time which an electriccontrolling circuit allows the weighted ball slide to apply force to theneedle.

According to the needle-punch method, a dielectric is advantageouslyselected so as to be amenable to puncture. That is, a polymer dielectrichaving a predetermined softening point may be selected, and it may beadvantageous to heat the needle or the stage upon which a sensor ismounted, or both, so as to soften the polymer dielectric duringpuncture. When the needle, stage, or both are heated so as to soften thepolymer dielectric during puncture, care should be taken such thatexcessive heat is not applied, which heat would cause adjacent sensorlayers to delaminate. Particularly, any heating should be carried out soas not to cause delamination between the dielectric and the electricallyconductive material.

Materials suitable for use as dielectric 40 according to theneedle-punch method of the present invention may comprise any of avariety of compounds, for example polymer compounds which adhere well toconductive strips 34, 36, and 38, especially during and after the needlepunching step. Suitable polymers may be selected from those in the alkydresin family (unsaturated polyester resin) and may contain phthalicanhydride, maleic anhydride, various glycols, and may additionallycontain unsaturated oils. Other suitable polymer precursors fordielectric formation are siloxane copolymers or siloxane-imidecopolymers. A preferred material is available as part number ESL 240 SBfrom Electro Science Laboratories in King of Prussia, Pa.

According to the needle punch method described herein, as in the case ofthe laser hole or opening formation method, a plurality of holes 56 maybe formed in dielectric 40 defining a plurality of working electrodes154, and a variety of shapes and sizes of hole or opening 56 may becreated. For example, a needle may be oriented at an angle relative todielectric layer 40 other than a perpendicular angle during punching, ormay be moved relative to the dielectric layer during hole or openingformation. Thus, oval-shaped, elongated, or line-shaped workingelectrode or electrodes 154 may be formed.

Due to the physical disruption of dielectric layer 40 and, in someinstances, the underlying electrically conductive strip 38 inherent inthe laser hole and needle punch hole formation methods, theabove-described electrochemical delamination analysis may beadvantageously carried out after formation of holes or openingsaccording to those methods.

Referring now to FIGS. 3 and 4, embodiments of the present invention areshown in which electrolyte 58, membrane 60, gasket 66, and cover member64 are illustrated. FIG. 3 is a cross-sectional view taken through line3--3 of the partial assembly of FIG. 1, illustrating additionalcomponents, and FIG. 4 is a cross-sectional view taken through line 4--4of the partial assembly of FIG. 2, also illustrating similar additionalcomponents. Specifically, provided atop reference electrode 50, counterelectrode 52, and working electrode 54 or 154 in a preferred embodimentis electrolyte 58. Electrolyte 58 may be deposited in any manner so asto contact the above-noted electrodes without contacting conductivestrips 34, 36, and 38 outside of the electrode region. That is, withreference to FIG. 1, electrolyte advantageously covers open printedregion 48 entirely but does not extend outside of the region which iscovered by dielectric layer 40 in the direction of the first end 41 ofthe substrate, that is, does not contact conductive strips 42, 44, or 46outside of the region in which they are selectively exposed to defineelectrodes.

Electrolyte 58 preferably comprises a gel or solid-state electrolytethat swells to less than 2 times its dry volume when contacted withwater or water vapor. More preferably, a solid-state electrolyte with aswell value of from about 5% to about 25%, most preferably from about 5%to about 10%, and which allows ion transport and does not allow electrontransport, is utilized. A swell value of 10% is understood to indicatean increase in volume over a dry volume of an additional 10% whencontacted with water or water vapor.

In a preferred embodiment, electrolyte 58 is a solid-state, chemicallyor physically cross-linked polymer that may be deposited from an organicsolvent, that is hydrophilic in the solid state, that has an oxygenpermeability of at least 3 Barrers, and that adheres well to surfaces towhich it is applied, that is, dielectric 40 and electrodes 50, 52, and54 or 154. Electrolyte 58 is also advantageously stable to any chemicalspecies or reaction product generated during the operation of thesensor.

A Barrer is a unit of gas permeability defined by gas flux (at standardtemperature and pressure) per unit time, multiplied by the thickness ofthe material, and divided by the area of the material and the pressuredifferential across the material in that area. The Barrer is defined byEquation 1. ##EQU1##

Permeability values in Barrer units may be obtained according to thefollowing method. A membrane to be tested is mounted so as to becontacted on a first side by flowing water having a partial pressure ofoxygen equal to the partial pressure of oxygen in air, and contacted ona second side opposite the first side with flowing carrier gas,specifically nitrogen. The areas on the first and second sides of themembrane contacted by water and carrier gas, respectively, are of equaldimension. The carrier gas flowing past the membrane is analyzed foroxygen content using a potentiometric palladium oxygen sensor. Usingmeasured values of oxygen flux as a function of time, the area of themembrane contacted by water on the first side and by carrier gas on thesecond, the thickness of the membrane, and the oxygen partial pressuredifferential across the membrane, permeability values in Barrer unitsaccording to Equation 1 are determined.

Preferably, electrolyte 58 is a solid-state ion transport electrolyte,specifically a cation or anion exchanger in its porotic form orexchanged with a metal ion. Such solid-state ion transport electrolytesare known in the art and suitable examples include a material sold underthe trademark Nation®, manufactured by DuPont and available from AldrichChemical Company of Milwaukee, Wis. In the preferred embodiment,lithium-exchanged Nafion is employed, but sodium-exchanged Nafion orporotic Nafion may also be utilized. Preparation of such exchangedNafion is well-known to those of ordinary skill in the art. CertainNafion compounds suitable for use as an electrolyte in the presentinvention are described in U.S. Pat. No. 4,536,274, referenced above.

Generally, electrolyte 58 is formed over the electrode region byapplying lithium-exchanged Nafion in a solvent carrier to the electrodesand allowing the solvent to evaporate. It may be advantageous to heatthe resultant residue for a period of time to completely evaporate thesolvent carrier in a curing process. The electrolyte may be applied tothe electrode area by spin-casting, a well-known technique in the art.Devices for carrying out spin-casting are readily commerciallyavailable.

Atop and completely covering electrolyte 58, semipermeable membrane 60is provided in the preferred embodiments illustrated in FIGS. 3 and 4.Membrane 60 serves the purposes of protecting electrolyte 58 andelectrodes 50, 52, and 54 or 154 from species such as metal ions,battery metals and liquid water which may be harmful to their intendedoperation, while passing species such as water vapor and oxygennecessary for sensor operation. Membrane 60 further serves the purposeof limiting passage of oxygen to the working electrode 54 or 154 so asto enhance the non-depleting nature of the sensor. A membrane suitablefor use in the present invention has an oxygen permeability less thanabout 8 Barrers, preferably from about 0.1 to about 5 Barrers, and morepreferably from about 0.2 to about 3 Barrers.

Membrane 60 is also preferably rapidly permeable to water vapor whenNafion is used as an electrolyte as, according to that embodiment, wetupof Nafion is necessary for its operation. Additionally, sensorstabilization is effected upon saturation of membrane 60 with watervapor. Thus, rapid permeability of membrane 60 to water vapor lessensthe time to sensor stability in that regard. Preferably, membrane 60 hasa water vapor permeability of at least 3 Barrers. Membrane 60 is alsopreferably impermeable to liquid water, and as a result impermeable toions. A membrane deposited so as to have an impedance across themembrane, measured between the counter electrode on one side of themembrane and another electrode in saline solution on the other sidethereof, of at least 12 megohms may be desirable in this regard. It isto be understood that the precise impedance across the membrane is notcritical to the invention, but that impedance across the membrane maysimply be used to evaluate materials which may be suitable for use inthe present invention.

A membrane suitable for use in the present invention desirably has adegree of flexibility that will allow a solid-state electrolyte to swelland contract according to the values noted above, while adhering toadjacent sensor layers. Additionally, a membrane which is too brittlewill delaminate from underlying sensor layers. The membrane alsodesirably possesses a durability sufficient to withstand pressure froman overlaying gasket according to a preferred embodiment of the presentinvention described below, while maintaining desirable microscopicphysical and chemical characteristics.

Glass transition temperature may serve as an indicator of flexibilitydesirable in a material suitable for use as a membrane according to apreferred embodiment of the present invention. As is the case formembrane impedance, glass transition temperature of the membrane is notcritical to the invention, but may be used as an evaluation method ofselecting advantageous membranes. Material used for membrane fabricationshould not have a glass transition temperature at or near thetemperature at which the sensor normally operates, that is, 37° C.according to common blood analysis techniques. Preferably, membrane 60is fabricated from a material having a glass transition temperature offrom about -40° C. to about 110° C. but not at sensor operationtemperature. More preferably, the glass transition temperature ofmembrane 60 is from about -15° C. to about 85° C. but not at sensoroperation temperature.

Durometer values may indicate durability characteristics desirable in amaterial suitable for use as a membrane according to a preferredembodiment of the present invention. Material selected for fabricationof membrane 60 should have a Shore A hardness higher than that of thegasket, typically at least about 20, preferably at least about 50, andmore preferably at least 70. The stated values are measured according tothe well-known ASTM standard procedures.

As is the case with respect to other components of the inventive sensoras described above, initial evaluation of suitable membrane materialsmay be made on the basis of electrochemical investigation.

Specifically, in a sensor fabricated according to embodimentshereinabove described, the working and reference electrodes may bebiased at a predetermined potential, and the sensor may be exposed to anaqueous solution to determine the water vapor permeabilitycharacteristic of the membrane. The membrane desirably passes watervapor rapidly, resulting in a current output which rapidly equilibratesto a steady-state reading, and remains relatively stable as a functionof time.

The oxygen permeability of a membrane may also be tested according tothe above-noted electrochemical methods, by exposing such a sensor to asolution having a known, predetermined PO₂, polarizing the sensor asdescribed above, and measuring current output. A relatively low andstable output current may signify that the membrane operates, in concertwith the small working electrode described above, to provide asubstantially non oxygen-depleting sensor.

Additionally, the sensor may be exposed to a solution containingcontaminants which, if they were to permeate the membrane, wouldadversely affect the electrolyte and/or electrodes or beelectrochemically oxidized or reduced, giving false test result readingsor no reading at all. Thus, exposure of the sensor to such a solution,and measurement of a polarogram during or following such exposure maydetermine permeability characteristics of a membrane. The potentiallongevity of a sensor may be tested by carrying out such electrochemicaltesting over a period of days and/or months where the sensor is storedexposed to such test solution and potentially biased. As noted above,according to a preferred embodiment of the present invention, anypolarogram taken is advantageously free of significant peaks in apotential range in which electrochemical measurements are taken. In thepreferred embodiment of the present invention, this is at approximately-0.800 volts, ±0.300 volts vs. silver/silver chloride during its usefullife.

An additional electrochemical test of significance is to expose such asensor to a solution having a known, predetermined PO₂ for a long periodof time, the sensor being polarized as described above, and observingthe current output as a function of time. A desirable membrane oxygenpermeability is such that, in a sensor having a small working electrode,exposure of the sensor to a sample having a pO₂ as low as that whichwill likely be measured and exhibits a current output which is as low aspossible but high enough that pO₂ may be determined within an acceptablemargin of error. A current output which is substantially free oflong-term drift is also desirable. As used herein, "free of long-termdrift" may be defined by current output, at a temperature stable to±0.1° C., at stable pO₂, and at a constant potential, which fluctuatesless than ±10% over a 24 hour period.

As noted, membrane 60 is desirably impermeable to water, permeable towater vapor and of limited permeability to oxygen. Therefore, themembrane is formulated to contain pendant groups which cause the polymerchains to interact in a way that imparts desirable physicalcharacteristics to the membrane, and which provide the overall membranecomposition with a desirable degree of polarity.

Generally, a membrane is water permeable if it contains a sufficientnumber of groups having a sufficient degree of polarity, especially ifit contains groups which are amenable to hydrogen bonding. Generally, amembrane is gas permeable if it contains a sufficient degree of freevolume. Free volume is decreased by such factors as polymer chaincross-linking and/or the presence of pendant groups in polymer chainsthat cause the polymer chains to bind to each other tightly. Anadditional consideration for gas permeability is the polarity of theparticular gas molecule relative to the polarity of the membranepolymer. A polymer having greater numbers of groups having a high degreeof polarity will generally be more permeable to a gas made up of polarmolecules.

Typically, membrane 60 comprises a copolymer containing one or moretypes of pendant side groups selected, and occurring with a degree offrequency, so as to provide inter-chain dipole-dipole interactions thatcreate a proper degree of free volume within the polymer and whichprovide the polymer with a proper degree of polarity. Additionally, suchselection provides the polymer with desired overall mechanicalcharacteristics such as a proper degree of flexibility. The pendant sidegroup or groups are thus selected so as to have a polarity great enoughto limit oxygen permeability to a level which provides for anon-oxygen-depleting oxygen sensor, and which allows for rapid watervapor transport, but which is low enough that the polymer remainshydrophilic, and is thus not permeable to ions. Providing a polymer forfabrication of a membrane that satisfies these requirements and isimpermeable to carbon dioxide requires a balancing between polaritywhich decreases free volume, thus decreasing gas transport, and polaritywhich is attractive to carbon dioxide molecules.

According to one embodiment of the present invention, membrane 60comprises a random or block copolymer comprising the polymerizationproduct of at least one nitrile-containing monomer such as an alpha,beta-ethylenically unsaturated nitrile, for example acrylonitrile ormethacrylonitrile and at least one conjugated diene monomer.Acrylonitrile is preferred for use as a nitrile-containing monomeraccording to a preferred embodiment. Non-limiting examples of conjugateddienes which may be used include 1,3-butadiene; isoprene;2,3-dimethyl-1,3-butadiene; 1,3-pentadiene; 3-butyl-1,3-octadiene andothers well-known to those of ordinary skill in the art, and mixturesthereof, with 1,3-butadiene being preferred.

As used herein, the term "polymerization product" refers to a product ofstandard polymerization methods such as anionic polymerization, cationicpolymerization, free radical polymerization, coordinationpolymerization, and the like.

Preferably, a random copolymer comprising the above-noted polymerizationproducts is formulated, in which the nitrile-containing monomer is addedin an amount of from about 5% to about 80%, preferably from about 15% toabout 55%, and more preferably from about 30% to about 40% by weight,based on the weight of the polymerization product. While a randomcopolymer is generally selected and formulated, a block copolymercomprising polymerized blocks of nitrile-containing monomers andconjugated diene monomers may be selected and formulated.

A suitable membrane material desirably comprises polymer chains whichare not cross-linked or, if cross-linked to any degree, maintain adesirable glass transition temperature and a desirable oxygenpermeability.

A polymer for use in fabricating a semipermeable membrane in accordancewith one embodiment of the present invention is a randomacrylonitrile-butadiene copolymer containing acrylonitrile in an amountof from about 5% to about 80% by weight, preferably from about 15% toabout 65% by weight, more preferably from about 30% to about 50% byweight. The acrylonitrile may be isotactic, syndiotactic, or atactic.Generally, the acrylonitrile is atactic. The butadiene may be of avarious degree of isometric purity. Typically, the butadiene group isnot pure isomerically but is a mixture of cis and trans-monomers andincludes pendant vinyl groups. The butadiene group may be saturated orunsaturated, and is generally substantially unsaturated in a preferredembodiment.

According to another embodiment, membrane 60 comprises theabove-described nitrile/conjugated diene copolymer to which thepolymerization product of a vinyl halide, a vinylidene halide, either ofthe two copolymerized with a nitrile-containing monomer, or acombination of any of the above is added in an amount of from about 1 toabout 70% by weight, based on the weight of the overall membranecomposition, preferably in an amount of from about 15% to about 50% byweight, and more preferably in an amount of from about 30% to about 40%.Preferably isotactic, syndiotactic or atactic polyvinyl chloride or arandom copolymer of polyvinylidene chloride and acrylonitrile in whichthe polyvinylidene chloride is added in an amount of from about 40% toabout 95% by weight, preferably from about 60% to about 90%, and morepreferably about 80% by weight, based on the weight of thepolyvinylidene chloride/acrylonitrile copolymer, are selected and added.The addition of these species to the nitrile/conjugated diene polymerhas the effect of lowering the oxygen permeability of the membrane, andresultant normalized current response of the sensor. Additionally,addition of these species typically provide a membrane which is moredurable than a nitrile/conjugated diene polymer alone, which isadvantageous in the fabrication of a sensor having a sample chamberconstructed on the membrane, that is, a sample chamber defined in partby the membrane, and constructed so as to expose the membrane, above theelectrode area, to samples to be analyzed. Additionally, addition ofthese species may enhance sensor longevity. Thus, the membrane may betailored with respect to oxygen permeability and durability by adjustingthe composition ratio of the polymer blend.

The polyvinyl chloride and/or polyvinylidene chloride/acrylonitrilecopolymer is preferably incorporated in the nitrile/conjugated dienecopolymer to form a mixed or blended polymer composition by conventionalblending technique such as cosolvation in a mutual solvent, standardblending processes, and the like. In a preferred embodiment, cosolvationis employed.

One of the advantages of cosolvation of a polyvinylidenechloride/acrylonitrile copolymer with a nitrile/conjugated dienecopolymer is that the miscibility of the two copolymers in the mixture,and thus the homogeneity of the mixture, is increased.

According to another embodiment, membrane 60 is made from a polymer orcopolymer of an ester of an alpha, beta-ethylenically unsaturatedcarboxylic acid, for example a member of the group of alkyl acrylates ormethacrylates. Optionally, there may be included in the copolymer one ormore monomers selected from the group including vinyl acetate, styrene,vinyl toluene, vinyl chloride, vinylidine chloride, and an alpha,beta-mono-ethylenically unsaturated acid or an amine-containing monomer.Typically, membrane 60 comprises the polymerization product of at leastone acrylate monomer, that is, a monomer having the formula CH₂═C(R₁)(COOR₂), where R₁ and R₂ are each selected from the groupconsisting of hydrogen, hydrocarbon groups, and alcohol groups and R₁and R₂ can be the same or different. Hydrocarbon groups such ashydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl,and the like may be selected. As used herein, the terms "hydrocarbon","alkyl", "cycloalkyl" and similar hydrocarbon terminology is meant toinclude alcohols and hydrogen, although specific reference to theinclusion of hydrogen and/or alcohols is frequently made herein.Examples of such groups are methyl, propenyl, ethynyl, cyclohexyl,phenyl, tolyl, benzyl, hydroxyethyl and the like. R₁ is preferablyselected from groups including hydrogen and the general class of loweralkyl compounds such as methyl, ethyl, or the like.

R₂ is preferably an alkyl group, preferably having 1 to 24 carbon atoms,most preferably 1 to 18 carbon atoms; and alkenyl group, preferablyhaving 2 to 4 carbon atoms; an aminoalkyl group, preferably having 1 to8 carbon atoms, and optionally substituted on the nitrogen atom with oneor, preferably two alkyl groups, preferably having 1 to 4 carbon atoms;an alkyl group, preferably having 1 to 4 carbon atoms, having a five orsix-membered heterocyclic ring as a substituent; an allyloxyalkyl group,preferably having up to 12 carbon atoms; an alkoxyalkyl group,preferably having a total of 2 to 12 carbon atoms; an aryloxyalkylgroup, preferably having 7 to 12 carbon atoms; an aralkyl group,preferably having up to 10 carbon atoms; or a similar alkyl or aralkylgroup having substituents which will not interfere with thepolymerization of the ester.

That is, homopolymers and copolymers advantageously used for membrane 60may include esters selected from the group consisting of (C₁ -C₂₄)alkylesters of acrylic acid, preferably a (C₁ -C₄)alkyl acrylate, di(C₁-C₄)alkylamino(C₂ -C₄)alkyl esters of acrylic acid, (C₁ -C₈)alkoxyalkylesters of acrylic acid, (C₆ -C₁₀)aryloxyalkyl esters of acrylic acid,(C₇ -C₁₀)aralkoxyalkyl esters of acrylic acid, and (C₇ -C₁₀)aralkylesters of acrylic acid. The copolymers of this invention includepolymers in which more than one monomer is selected from a given group,for instance, the case where the polymer is a copolymer of at least two(C₁ -C₂₄)alkyl acrylates. Other copolymers of the invention comprisemonomers which may or may not be acrylates, such as copolymers of atleast one (C₁ -C₂₄)alkyl acrylate and at least one other copolymerizableethylenically-unsaturated monomer. This copolymerizable monomer may beacrylonitrile or dimethylaminoethyl acrylate, preferably when the alkylacrylate is a (C₁ -C₂₄)alkyl acrylate.

Among the esters embraced by the formula CH₂ ═C(R₁)(COOR₂) which aresuitable monomers are unsubstituted alkyl acrylates, in which the alkylgroup can have branched or straight-chain, cyclic or acyclic spatialconfigurations, such as methyl acrylate, ethyl acrylate, propyl,isopropyl and cyclopropyl acrylates, isobutyl, t-butyl, n-butyl andcyclobutyl acrylates, pentyl and cyclopentyl acrylates, hexyl andcyclohexyl acrylates, heptyl and cycloheptyl acrylates, octyl,acrylates, including 2-ethylhexyl acrylate, nonyl acrylates, decylacrylates, undecyl alcrylates, lauryl acrylates, myristyl acrylates,cetyl acrylates, stearyl acrylates, and the like; aralkyl acrylates,such as phenylethyl acrylates, phenylpropyl acrylates, and the like;aralkyl acrylates, in which the aryl group is substituted with alkylgroups, halogen atoms, alkoxy groups, nitro groups, or similarsubstituents which will not interfere with the polymerization reaction;alkenyl acrylates, such as allyl acrylate, and the like; aminoalkylacrylates, such as dimethylaminoethyl acrylate, phenylaminoethylacrylates, t-butylaminoethyl acrylates, dimethylaminobutyl acrylates,diethylaminoethyl acrylate, and the like; alkyl acrylates having aheterocyclic group as a substituent on the alkyl group, such asmorpholinoalkyl acrylates, oxazolidinylalkyl acrylates, piperidinodalkylacrylates, dioxolanylalkyl acrylates, i.e., ketals and acetals ofglyceryl acrylate, and the like; iminoalkyl acrylates, such asketiminoalkyl acrylates and aldiminoalkyl acrylates; alkoxyalkyl,aryloxyalkyl, and aralkoxyalkyl acrylates, such as methoxyethylacrylate, ethoxyethyl acrylate, butoxyethyl acrylates, hexyloxypropylacrylates, ethoxypropyl acrylates, propoxybutyl acrylates, hexloxyhexylacrylates, phenoxyethyl acrylates, benzyloxyethyl acrylates, and thelike; and allyloxyalkyl acrylates, such as allyloxyethyl acrylate,allyloxyethoxyethyl acrylate, allyloxypropyl acrylate, and the like. Bisacrylate esters of diols, such as the diester of 1,4-butanediol andacrylic acid, can also be used. Other esters of acrylic acid which donot contain substituents which would interfere with the polymerizationof these esters are also suitable.

R₂ is preferably selected from linear, branched and cyclic hydrocarbonsand alcohols of from 1 to 20 carbon atoms. R₁ and R₂ may eachindividually, or both, comprise groups having substituted fornon-substituted hydrocarbons and may comprise heteroatoms. Preferably,any substitution results in non-reactive groups.

A preferred class of polymer mixtures suitable for fabrication ofmembrane 60 includes the above-noted polymer or copolymer of at leastone acrylate monomer which may be polymerized with at least oneadditional monomer. A non-limiting list of suitable additional monomerswhich may be polymerized with one or more acrylates to formpolymerization products include at least one nitrile-containing monomer;the polymerization product of at least one monomer having the formulaCH₂ ═C(R₁)(CONR₂ R₃), where R₁, R₂, and R₃ are each selected from thegroup consisting of hydrogen, hydrocarbon groups, and alcohol groups andR₁, R₂, and R₃ can be the same or different; and the polymerizationproduct of at least one monomer having the formula CH₂ ═C(R₁)(OCOR₂),where R₁ and R₂ are each selected from the group consisting of hydrogen,hydrocarbon groups, and alcohol groups and R₁ and R₂ can be the same ordifferent.

According to one preferred class of polymer mixtures suitable forformulation of membrane 60, a mixture is selected which includes thepolymerization product of at least one monomer having the formula CH₂═C(R₁)(COOR₂) where R₁ is selected from the group consisting of hydrogenand lower alkyl groups, R₂ is selected from the group consisting oflinear, branched and cyclic hydrocarbons and alcohols of from 1 to 20carbon atoms, R₁ and R₂ being the same or different, present in anamount of from about 20% to about 80% by weight based on the weight ofthe membrane, preferably from about 40% to about 60% by weight. Alsoincluded is the polymerization product of at least onenitrile-containing monomer present in an amount of from about 15% toabout 80%, preferably from about 20% to about 30% by weight, and thepolymerization product of either a monomer having the formula CH₂═C(R₁)(OCOR₂) where R₁ and R₂ are each selected from the groupconsisting of hydrogen, lower alkyl groups and lower alcohols and R₁ andR₂ can be the same or different, or a monomer having the formula CH₂═C(R₁)(CONR₂ R₃) where R₁, R₂, and R₃ are each selected from the groupconsisting of hydrogen and lower alkyl groups and R₁, R₂, and R₃ can bethe same or different. A combination of the latter two monomers may beadded, and in a preferred embodiment the monomer having the formula CH₂═C(R₁)(OCOR₂), where R₁ and R₂ are each selected from the groupconsisting of hydrogen, lower alkyl groups and lower alcohols and R₁ andR₂ can be the same or different and is present in an amount of fromabout 15% to about 30% by weight.

Specific examples of monomers suitable for polymerization to form amembrane copolymer composition according to this embodiment of thepresent invention include, but are not limited to: acrylonitrile,2-ethylhexylmethacrylate, methylmethacrylate, dodecylmethacrylate,vinylacetate, cyclohexylmethacrylate, 2-hydroxypropylmethacrylate, andacrylamide.

A random copolymer of components described above may be formulated, or ablock copolymer comprising blocks having molecular weights of from about10,000 to about 100,000 of the above-described monomers may beformulated. Preferably, the membrane is formulated from a randomcopolymer of the above-noted monomers. The copolymer may becross-linked, but is preferably not cross-linked or cross-linked to alimited extent so as to maintain adequate free volume and flexibility.

The polymer membrane compositions of the invention may be produced byany convenient polymerization method, such as anionic polymerization orfree-radical polymerization, or the like.

According to another embodiment, membrane 60 comprises a polyimidecompound, which may be a homopolyimide, or a copolymer comprising imidefunctionalities and other groups. Preferably, according to thisembodiment, membrane 60 comprises aromatic polyimides of aromaticdiamines which are substituted in the nucleus by alkyl, and aromaticdiamines which carry sulfonic acid groups which may be in salt form, andaromatic tetracarboxylic acids. In a preferred embodiment, membrane 60comprises aromatic copolyimides of aromatic tetracarboxylic acids andtricarboxylic or aromatic tricarboxylic acids and a first aromaticdiamine and a second aromatic diamine which carries-SO₃ H--groups insalt form, said first and/or said second diamine being C₁ -C₄alkyl-substituted in both ortho-positions to at least one amino group.Such materials are described in U.S. Pat. No. 5,145,940, incorporatedherein by reference.

According to yet another embodiment, membrane 60 may comprise apolyamide, copolyamide or copolyimide-amide compound, preferablyaromatic copolyamides and copolyimide-amides of two aromatic diamines,one of which may contain sulfonic acid groups. Such compounds aredisclosed in European Patent Application No. EP-A-0473541, incorporatedherein by reference.

According to this embodiment, membrane 60 preferably comprises acopolyamide or copolyimide-amide of (a) at least one aromaticdicarboxylic acid radical of 8 to 20 carbon atoms and/or (b) at leastone trivalent aromatic tricarboxylic acid radical of 9 to 20 carbonatoms, each of which radicals is unsubstituted or substituted byhalogen, nitro, C₁ -C₄ alkyl or C₁ -C₄ alkoxy, (c) at least one firstdivalent and/or trivalent mononuclear or binuclear aromatic diamineradical and (d) at least one second divalent and/or trivalent aromaticmononuclear or binuclear diamine radical containing at least one --SO₃ Mgroup, each of which radicals is unsubstituted or substituted by halogenor C₁ -C₄ alkyl, and M is H⁺, a monoto trivalent metal cation, NH₄ ⁺ oran organic ammonium cation of 1 to 30 carbon atoms. When membrane 60comprises a copolyamide or copolyimide-amide containing --SO₃ M groups,such groups are desirably present to an extent less than that whichwould cause the membrane to be water permeable, and thus ion permeable.

M in the SO₃ M group as ammonium cation may be NH₄ ⁺ or an ammoniumcation of a primary, secondary or tertiary open-chain amine containingpreferably 1 to 24 carbon atoms, most preferably 1 to 16 carbon atoms,or an ammonium cation of a monocyclic or bicyclic secondary or tertiaryamine or of a tricyclic tertiary amine containing preferably 4 to 12carbon atoms.

M as a metal cation is selected in accordance with the teachings hereinregarding species which may not be desirable for incorporation intocomponents of the sensor 30. That is, mobile battery metals present inthe membrane are undesirable. With this in mind, M as a metal cation isdesirably a mono to trivalent cation of metals of the main groups andsubgroups, of the transition metals and of the noble metals. Mono ordivalent cations are preferred.

In a preferred embodiment of the invention, M is H⁺, NH₄ ⁺, an alkalimetal cation or a primary, secondary, tertiary or quaternary ammoniumcation of 1 to 24 carbon atoms.

The copolyamide or copolyimide-amide preferably contains 60 to 95 mol %,most preferably 75 to 95 mol %, of diamine radicals (c) and 5 to 40 mol%, most preferably 5 to 15 mol % of diamine radicals (d), based on saiddiamine radicals. The inherent viscosity of the copolyamides,copolyamide-imides or of the copolymers can be from 0.2 to 3.0 dl/g,preferably 0.3 to 2.0 dl/g and, most preferably, 0.3 to 1.2 dl/g.

Referring now to FIGS. 3-5, a preferred embodiment of the presentinvention is illustrated in which a sample chamber 62 is provided abovethe electrode region at end 43 of sensor 30 or 130. Chamber 62 isdefined by a substantially oval recessed region of cover member 64, theperimeter of which is held firmly against gasket 66 at seal 67, which isin turn held firmly against dielectric layer 60 at seal 69. Thus, samplechamber 62 is defined by dielectric layer 60, gasket 66 and cover member64. At least one passage 68, and preferably two passages 68 and 70,serving as inlet and outlet channels, respectively, are formed in covermember 64 to allow passage of a fluid sample such as blood into and outof the sample chamber 62 of the sensor. In the embodiment illustrated,channels 68 and 70 pass through cover member 64. However, channels maybe formed in any manner so as to provide a passageway through which afluid sample could reach sample chamber 62. For example, channels couldbe formed in gasket 66 between gasket 66 and cover member 64, betweendielectric 60 and gasket 66, etc.

Cover member 64 may be fabricated from any material that is unreactivewith a sample which passes into sample chamber 62 during analysis, asfor example, glass, ceramic, stainless steel, or plastic. Preferably,plastic is used in the formation of cover member 64.

Gasket 66 is advantageously formulated from a material which, when heldfirmly between cover member 64 and dielectric layer 60, forms a sealaround sample chamber 62 through which the passage of fluid and gas issubstantially barred such that testing may be carried out for a periodof at least 2 days under normal sensor operation. "Normal sensoroperation", as used herein, refers to operation in which a sensor isexposed to at least 10 blood samples a day and/or calibrators, qualitycontrol solutions, controls, proficiency testing solutions, calibrationverification materials, and the like while continuously polarized at 37°C. so as to measure a current response, or operation in which a sensoris exposed to a blood-mimicing saline solution and is polarized atmeasuring potential, continuously, as described above. Additionally,normal sensor operation may be defined by the test protocol describedbelow in Example 58. In actual use of a sensor according to the presentinvention, the sensor would not be polarized at a measuring potentialcontinuously. Therefore, it is to be understood that "normal sensoroperation" may refer to a prescribed longevity or durability measurementor both. The term "blood", as used herein, refers to whole bloodcontaining anticoagulants, plasma, serum or other blood solutions orsuspensions.

Typically, gasket 66 is formulated from a durable organic polymer whichdoes not creep or flow when stressed, which has a low durometer ratingso that damage to membrane 60 is minimized or eliminated, which is gasimpermeable, and which may be slightly hygroscopic and thus may swellslightly in the presence of solution containing water, to form efficientseals 67 and 69.

Preferably, material used in the fabrication of gasket 66 has a hardnessof between 10 and 100 on the Shore A scale, more preferably a hardnessof from about 40 to about 70 on the Shore A scale, and most preferably ahardness of from about 45 to about 55 on the Shore A scale.

Gasket 66 desirably has sub-microscopic properties which make it gasimpermeable, and is thus preferably formulated from a precursor having asufficient degree of unsaturated carbon-carbon bonds to form asufficiently highly cross-linked polymer compound when cured, or haveother means of attaining such a degree of cross-linking. Specifically,the material from which gasket 66 is made has an oxygen permeability ofless than about 20 Barrers, preferably less than about 5 Barrers, andmost preferably less than about 0.5 Barrers.

As gasket 66 is typically an organic polymer, it is fabricated so as notto contain a substantial amount of any mobile extractable materials suchas plasticizers which may leach into semipermeable membrane 60. Suchleaching of extractables can affect the microscopic physical propertiesof the membrane, disadvantageously effecting a change in the above-notedadvantageous permeability characteristics of the membrane. This is anespecially notable consideration with respect to sensors designed forlong-term use, on the order of for example days or months and to sensorsoperating with small test sample volumes. Additionally, as is the casefor other sensor components as described above, it is important thatmaterial selected for formation of gasket 66 be free of any specieswhich could migrate into a sample in chamber 62, affectingelectrochemical measurements, and/or destroying sensor components.Material used in the formation of gasket 66 is preferably selected to beessentially free of mobile transition and main group metals, especiallybattery metals such as iron, cobalt, nickel, lead, copper, extractables,and species such as sulfides which are deleterious to preferredelectrode materials, such that electrochemical response is not affectedover long-term sensor use, specifically for at least 2 days of normalsensor operation.

Gasket 66 is typically formed from a highly cross-linked elastomericcompound. Any elastomeric material which meets all the purity andphysical requirements listed above may serve. A preferred embodiment ofthis material is composed of a copolymer of an epoxy compound and asmall amount of another monomer which provides sites of unsaturation inthe copolymer. The second monomer may be added in amounts to give fromabout 0.1 to about 20% crosslinking upon cure (1% crosslinking wouldindicate that, on the average, 1 out of every 100 monomers in a chainwould be a point at which the elastomer is crosslinked). A morepreferred degree of crosslinking would be from about 1 to about 15% andthe optimum performance would be obtained in a material with from about6 to about 14% crosslinking.

A more preferred embodiment is obtained when the epoxy compound iscomposed of epichlorohydrin and the second monomer is allyl glycidylether. The sites of unsaturation provided by the second monomer allowthe material to be crosslinked to form an elastomer using any convenientfree radical mechanism, for example via peroxide.

In order to provide the proper amount of swelling in aqueous or bloodbased solutions, a third monomer may be added to the prepolymer mixture.Examples of suitable monomers include allyl alcohol, crotyl alcohol,methylvinyl carbinol, cinnamyl alcohol, ethylene glycol, propyleneglycol, 1,3-propanediol, glycerol, pentaerythritol, acrylamides, acrylicmonomers, and the like.

In addition, it may be necessary to add processing aids to theprepolymer. These include alkali stearates, alkali oxides, and the like.

A partial list of curing agents include 2,5-bis(t-butylperoxy)-2,5-dimethylhexane, dicumyl peroxide, di-tert-butylperoxide, and dilaurate peroxide. Any peroxide curing agent whichgenerates free radicals at temperatures below 180° C. and can bedispersed in the prepolymer may be used as the curing agent.

A polyfunctional monomer may be used to facilitate the crosslinkingreaction during the cure. This monomer should be similar to theunsaturated sites in the prepolymer to react easily with these sites.Examples include 2-ethyl-2-hydroxymethyl-1,3-propanedioltrimethacrylate, diethylene glycol diacrylate, 1,4-butanedioldiacrylate, divinyl benzene, glyceryl propoxy triacrylate, anddipentaerythritol monohydroxypentaacrylate. The preferred monomer is2-ethyl-2-hydroxymethyl-1,3-propanediol trimethacrylate.

This polyfunctional monomer should be added in an equivalent amount tothe unsaturation sites in the prepolymer. Equivalence in this casemeaning the same number of reactive sites are in the prepolymer as arefound in the polyfunctional monomer. For example, if the polyfunctionalmonomer is trifunctional, there should be one of these monomers forevery three sites of unsaturation in the prepolymer.

Specific epoxy polymerization products preferred for use as gasketmaterials include: the polymerization product of an epichlorohydrinhomopolymer having a molecular weight of from about 2,000 to about1,000,000 and alkyl glycidyl ether, the ratio of the epichlorohydrinhomopolymer to alkyl glycidyl ether being from about 0.5% to about 20%by weight, available under the trademark Grechon 1100 from ZeonChemicals of Kolling Meadow, Ill.;2-ethyl-2-hydroxymethyl-1,3-propanediol trimethacrylate, available ascatalog number SR-350 from Sartomer Company of Exton, Pa.; potassiumstearate; calcium oxide; (2,5-bis(t-butylperoxy)-2,5-dimethylhexane,available under the trademark Varox DBPH-50 from R. T. Vanderbilt ofNorwalk, Conn.; and stearic acid.

According to the present invention, a sample chamber 62 of any size,including a very large chamber, may be fabricated. Fabrication of alarge sample chamber may be advantageous in some circumstances. As notedabove, however, in the field of electrochemical analysis of blood, it iscommonly desirable to perform as many analyses as possible on a verysmall volume of blood. Thus, according to a preferred embodiment of thepresent invention, it is desirable to fabricate sensor 30 or 130 with asample chamber 62 that is as small as possible. Using the novelmaterials and methods of the present invention, a sample chamber havinga volume of less than 10 μl, preferably from about 0.8 to about 3 ml,and more preferably from about 1 to about 2 μl, which sensor mayeffectively be utilized for a period of many days or months, ispossible.

A variety of shapes and configurations of components comprising sensor30 or 130 may be achieved using the well-known thick and thin-filmtechniques. U.S. Pat. No. 4,571,292, which is incorporated by referenceherein, discloses a variety of electrode configurations that may beadvantageous in some circumstances. Variation of configuration inaccordance with these references and other configurations easilyachieved by one of ordinary skill in the art are within the scope of thepresent invention.

With this in mind, and with reference to FIGS. 1-5, the followingnon-limiting preferred dimensional specifications of a sensor fabricatedin accordance with a preferred embodiment of the present invention aregiven.

Substrate 32 may be of any of a variety of shapes and sizes. Accordingto one specific preferred embodiment of the invention substrate 32 isfrom about 0.5 to about 2 cm long, preferably about 1.2 cm long and fromabout 0.2 to about 1 cm wide, preferably about 0.5 cm wide. According toanother specific embodiment, substrate 32 is from about 3.5 to about 7cm long, preferably about 5.0 cm long and from about 1.5 to about 3.5 cmwide, preferably about 2.5 cm wide. Substrate 32 is from about 0.1 toabout 0.5 mm thick, preferably about 0.25 mm thick. Conductive strips34, 36, and 38 are deposited each in a thickness of from about 0.002 toabout 0.04 mm thick, preferably about 0.013 mm thick. Conductive strips34 and 36, at end 43 of the sensor, are from about 0.5 to about 3.0 mmwide, preferably about 1.25 mm wide. Conductive strip 38, at end 43 ofthe sensor, is from about 0.02 to about 0.4 mm wide, preferably about0.10 mm wide.

Dielectric layer 40 is preferably deposited in a thickness of from about0.004 to about 0.05 mm thick, preferably about 0.019 mm thick. Thicknessvalues are given after firing or curing. Although dielectric layer 40abuts edges of conductive strips 34 and 36 in the embodimentsillustrated in FIGS. 1-4, such need not be the case. The open printedregion 48 defined by dielectric layer 40 preferably includes a gap 49having a width of from about 0.02 to about 0.4, preferably about 0.10mm.

Thus, the exposed surface area of reference electrode 50 and counterelectrode 52 is about 1.6 mm² for each, and working electrode 54 has asurface area of about 0.01 mm², according to the embodiment illustratedin FIG. 1. These exposed surface area dimensional specifications do nottake into consideration surface area due to the edges of the electrodes,defined by the thickness of the electrodes as deposited. Such edgedimensions are minimal relative to the overall electrode areas. However,the exposed surface area specifications are thus somewhat approximate.

According to the embodiment illustrated in FIGS. 2 and 4, in which alaser or a needle is used to form a hole or opening 56 in dielectriclayer 40 exposing working electrode 154, hole or opening 56 may beformed having a diameter of as small as 0.5 mm, and is typically formedin any size up to approximately 100 mm in diameter. Typically, hole oropening 56 has a diameter of about 3 mm. Thus, the surface area ofworking electrode 154 may be from about 2×10⁻¹³ m² to about 8×10⁻⁹ m².

Polymer electrolyte 58 is typically spun-cast over the electrodes anddielectric to a thickness of from about 0.001 mm to about 0.050 mm,preferably from about 0.005 mm to about 0.010 mm thick. During thespin-casting process, regions of conductive strips 34, 36 and 38 thatare not covered by dielectric 40, and contact pads 42, 44 and 46, aremasked. Accordingly to an alternate method, polymer electrolyte 58 maybe deposited in a dropwise manner from an X-Y-Z table such as thatdescribed above, to control placement.

Thereover, cover membrane 60 is deposited, preferably spun-cast to athickness from about 0.005 mm to about 0.050 mm, preferably from about0.010 mm to about 0.020 mm. As is the case for polymer electrolyte 58,cover membrane 60 may alternately be deposited in a dropwise manner froman X-Y-Z table. The dimensions of gasket 66 and cover member 64 are suchthat a sample chamber 62 has a width of from about 1 to about 10 mm,preferably from about 3 to about 4 mm and a height of from about 0.3 toabout 2.0 mm, preferably from about 0.6 to about 0.9 mm is defined. Asnoted, chamber 62 has a preferred volume of from about 1 to about 2 μl.

In the following examples, a Faraday cage was used when radio frequencysignal interference was noted. All fluid samples were held at a constanttemperature of at or near 37° C., physiological temperature.

The following examples are intended to illustrate the benefits of thepresent invention, but do not exemplify the full scope of the invention.For example, although the thick-film technique is exclusivelyexemplified, it is to be understood that the thin-film technique may beselected; although three-electrode sensors are exclusively exemplified,two-electrode sensors may be fabricated. It is also noted that manyfeatures of the invention, for example the novel membrane and gasketcompositions and the chamber construction and working electrodeformation, are not limited to use in a planar, solid-state oxygensensor. Additionally, as noted above, the specific shape and arrangementof the electrodes and conductive strips of the present invention may besignificantly altered, such alteration being within the scope of thepresent invention. Use of components of the present invention in anon-planar sensor, and/or one having a liquid electrolyte may beadvantageous, as well as use in sensors constructed to detect a varietyof analytes. Non-sensor use of the membranes, gaskets, and chamber,and/or electrode formation described herein is also within the scope ofthe present invention. These and other modifications and theirequivalents are understood to be within the scope of the presentinventions.

EXAMPLE 1

Referring now to FIGS. 1-5, a partial assembly of a planar oxygen sensor30 having substrate layer 32 and conductive metal strips 42, 44, and 46was fabricated in accordance with a method of the present invention on a2.5 cm by 5 cm electrically non-conducting substrate 32 comprisingapproximately 96% alumina and approximately 4% glass binder, availablefrom Coors Ceramic Company, Grand Junction, Colo. Using the thick-filmtechnique, conductive strip 34 was fabricated by silk-screening a 0.01mm emulsion silver paste available as part number 3571 UF from MetechCompany of Elverson, Pa. onto the substrate. A 325 mesh screen made of1.1 mil diameter SS wire was used. Subsequently, a 0.4 mil emulsionsilver/silver chloride paste, available as part number PC 10299 fromMetech, was silk-screened over a portion of conductive strip 34 at end43 of sensor 30, covering an area of conductive strip 34 at least aslarge as, and preferably larger than, the area of conductive strip 34 tobe exposed by open printed region 48 to define reference electrode 50. Ascreen similar to that described above was used. For purposes ofclarity, in FIGS. 3 and 4 reference electrode 50, created in accordancewith this example as a silver/silver chloride electrode, is illustratedas a single metal layer. In this and subsequent examples, a BTU 7 zonebelt furnace with a 3 zone dryer, from Fast Fire of Billerica, Mass. wasused in firing pastes. Firing was carried out per manufacturer'srecommendations, ramped to the peak conditions listed below. Conductivestrip 34 was fired at 850° C. for 10 minutes. Subsequently, a goldpaste, available as part number JM 1301 from Johnson-Matthey, of WestDeptford, N.J. was silk-screened onto substrate 32 to define conductivestrip 36. A similar emulsion and screening technique was employed. Thepaste was fired at 850° C. for 10 minutes. Then, a 0.2 mil emulsionhigh-purity platinum paste, as described above and available as partnumber PC 10208 from Metech, was silk-screened onto substrate 32 todefine conductive strip 38. A 400 mesh screen made of 0.9 mil diameterSS wire was used. The paste was fired at 870° C. for 13 minutes.

Substrate 32 was 0.25 mm thick. Conductive strips 34 and 36 weredeposited on substrate 32 so as to be 1.25 mm wide at end 43 ofsubstrate 32, and 0.014 mm thick. Conductive strip 38 was deposited soas to be 0.1 mm wide at end 43 of substrate 32. At end 41 of substrate32, where conductive strips 34, 36, and 38 define contact pads 42, 44,and 46, respectively, these strips were deposited so as to 0.25 cm wide.Dimensions given in this and subsequent examples are those after firing,and are approximate.

EXAMPLE 2

A partial assembly of a planar oxygen sensor 30 having substrate layer32 and conductive metal strips 42, 44, and 46 was fabricated inaccordance with Example 1 with the following exceptions. Substrate 32was approximately 11 mm long and approximately 4.5 mm wide. Contact pads42, 44, and 46 were each approximately 0.5 mm wide. Contact pads 42, 44,and 46 were each fabricated from gold, and portions of conductive strips34, 36, and 38 connecting the contact pads with the electrodes werefabricated from gold.

EXAMPLE 3

Referring now specifically to FIGS. 1 and 3, onto a partial assembly ofeach of the sensors fabricated in accordance with Examples 1 and 2, aceramic dielectric 40, available as part number 9615 from DuPont wassilk-screened as a 0.6 mil emulsion over a large portion of end 43 ofsensor 30. A 325 mesh screen made of 1.1 mil diameter SS wire was used.The silk-screening was effected in a manner so as to leave an openprinted region 48, exposing areas of conductive strips 34 and 36 of 1.25mm by 1.25 mm; an area of conductive strip 38 of 0.1 mm by 0.1 mm; andareas between the exposed areas of the conductive strips. Thus, openprinted region 48 exposes portions of conductive strips 34, 36, and 38and defines reference, counter and working electrodes 50, 52, and 54.Dielectric 40 was fired per manufacturer's recommendation, ramped to a750° C., 8 minute peak to give a thickness of about 0.019 mm. The areaof sensor covered by dielectric layer 40, extending from open printedregion 48, is not critical to the invention. The area need only extendover conductive strips 34, 36, and 38 away from open printed region 48to points that are not addressed by any electrolyte, as described below.

EXAMPLE 4

Referring now specifically to FIGS. 2 and 4, onto a partial assembly ofeach of the sensors fabricated in accordance with Examples 1 and 2, asensor 130 was fabricated in accordance with Example 3, with theexception that a polymer dielectric 40, specifically an unsaturatedpolyester resin containing phthalic anhydride, maleic anhydride, glycolsand unsaturated oils, available as part number ESL 240SB clear fromElectro Science Laboratories of King of Prussia, Pa., was deposited soas to completely cover conductive strip 38 in the sensor region at end43 of sensor 30. A 325 mesh screen made of 1.1 mil diameter SS wire wasused. Polymer dielectric 40 was cured at 200° C. for 2 hours in a curingoven available from Despatch, Inc. of Minneapolis, Minn. Dielectric 40was deposited so as to define a thickness of about 0.021 mm aftercuring. Thereafter, sensor 130 was positioned on an X-Y-Z table,available as model AUTOMOVE 302/201 from Asymtek Corporation ofCarlsbad, Calif. The X-Y-Z table was positioned such that conductivestrip 38 at end 43 of sensor 130 was under a needle, specifically partnumber PUN-W-.028-.750-10-.005, available from Small Precision Tools ofPetaluma, Calif. (12 micron tip radius). The needle was mounted on aweighted ball slide, having a down force adjustable by adding orremoving weight. The other side of the ball slide was fixed to the X-Y-Zpositioner. An electric probe was attached to the ball slide assembly,which probe was in contact with the needle and in contact with contactpad 46 of conductive strip 38. The X-Y-Z positioning table was heated soas to heat sensor 130 to approximately 65° C. to soften polymerdielectric 40. The needle was then lowered so as to puncture polymerdielectric 40 until the needle contacted conductive strip 38, closing anelectrical circuit, at which point the needle was immediately withdrawn.The electrical circuitry was controlled by computer software, easilyprogrammable by one of ordinary skill in the art. According to themethod of Example 4, a hole or opening 56 having a diameter of 0.050 mm,defining an electrode 38 having an area of 2×10⁻ 3 mm² was produced.

EXAMPLE 5

Referring now specifically to FIGS. 2 and 4, onto a partial assembly ofeach of the sensors fabricated in accordance with Examples 1 and 2, asensor 130 was fabricated in accordance with Example 3, with theexception that dielectric 40 was deposited so as to completely coverconductive strip 38 in the sensor region at end 43 of sensor 30, and adielectric material selected so as to substantially absorb radiationemitted from a Xenon laser was utilized. Specifically, a dielectricmaterial available as part number 9615 from DuPont was silk-screened andfired in accordance with Example 3. Subsequently, a Xenon laser wasfired at dielectric 40 to create a small hole or opening 56 aboveconductive strip 38, the hole or opening penetrating through dielectric40 to expose a small area of conductive strip 38, and defining workingelectrode 54. Hole 45 was formed between the exposed areas of conductivestrips 34 and 36 defining reference and counter electrodes 50 and 52respectively. A suitable laser is available as model number MEL-31 fromFlorod Corporation of Gardena, Calif. The exposed area of conductivestrip 38, defined by hole or opening 56, and defining working electrode54, was approximately circular with a diameter of approximately 2 mm.

EXAMPLES 6-8

Deposited atop the sensor region of sensors 30 and 130 fabricated inaccordance with Examples 3, 4, and 5, respectively, was solidelectrolyte 58. That is, Example 6 comprises a sensor fabricated inaccordance with Example 3 and including a solid electrolyte, Example 7comprises a sensor fabricated in accordance with Example 4 and includinga solid electrolyte, and Example 8 comprises a sensor fabricated inaccordance with Example 5 and including a solid electrolyte.

In each case, the electrolyte was deposited as lithium-exchanged Nafion,prepared from material available as Catalog Number 27,470-4 from AldrichChemical Company, by first neutralizing a 5% solid Nafion solution in acombination of isopropanol (10%), available as Catalog No. 27,470-4 fromAldrich, with lithium hydroxide to a pH of 7 and then concentrating thesolution under vacuum to a concentration of about 13.5% solids byweight. This solution was then spun-cast on the sensor assembly to givea film thickness of about 0.007 mm. During spin-casting, the contactpads were masked with tape. The lithium-exchanged Nafion electrolytefilm was then cured at 100° C. The film was deposited so as to contactreference, counter, and working electrodes 50, 52 and 54 or 154,respectively, without contacting other sensor components outside of thedielectric layer region.

EXAMPLES 9-14

Membrane materials were formulated from polyvinyl chloride (PVC) anddiundecylphthalate (DUP), by dissolving very high molecular weight PVC,available as Catalog No. 34676-4 from Aldrich Chemical Company andhaving an inherent viscosity of 1.02, and DUP, available as Catalog No.P-129 from Scientific Polymer Products, of Ontario, N.Y. intetrahydrofuran (THF) as approximately 15% solids by weight, andspin-casting the solution at approximately 1385 revolutions per minutefor ten seconds onto a surface. The THF solvent carrier was thenevaporated from the membrane by placing the sensor assembly in an ovenat 60° C. for two hours. Six membranes were made by this method with theweight percent of PVC in each example, based on the weight of the entiremembrane material, as follows: Example 9, 33%; Example 10, 50%; Example11, 67%; Example 12 73%; Example 13, 80%; Example 14, 83%.

The PVC/DUP membranes formulated in accordance with Examples 9-14 wereexamined to determine their permeability values. The results are plottedin FIG. 6. in which a plot of permeability as a function of percent PVCby weight of the polymer is displayed. The results indicate that at lowvalues of percent DUP by weight, good membrane permeability values arerealized but, as the percent by weight of DUP increases, permeabilityrapidly increased to non-optimum levels. Permeability of from 0.5 to 2.5Barrer units are preferred for use.

EXAMPLES 15-18

Membranes were formulated in accordance with the reaction method ofExamples 9-14 from mixtures of an acrylonitrile-butadiene (AB)copolymer, available as Part No. 533 from Scientific Polymer Products ofOntario, N.Y., and PVC as in Examples 9-14. Four AB/PVC membranes weremade by this method with the weight percent of PVC in each example,based on the weight of the entire membrane material, as follows: Example15, 0%; Example 16, 33%; Example 17, 50%; Example 18, 67%.

Membranes formulated in accordance with Examples 15-18 were tested todetermine their oxygen permeability characteristics, as in Examples9-14. Results are plotted in FIG. 6. It is notable that a wide range ofAB/PVC copolymer mixtures may be formulated within an optimumpermeability range. Thus, the blend may be formulated so as to satisfyboth oxygen permeability requirements and other physical and chemicalrequirements.

EXAMPLES 19-22

Membranes were formulated in accordance with the reaction method ofExamples 9-14 from a blend of the AB copolymer of Examples 15-18, andpoly(vinylidene chloride-co-acrylonitrile) (PVdC/AN), available ascatalog no. 396 from Scientific Polymer Products, having a molecularweight of 26,000. Four AB/PVdC/AN membranes were made by this methodwith the weight percent of PVdC/AN in these Examples, based on theweight of the entire membrane material, as follows: Example 19, 0%;Example 20, 33%; Example 21, 50%; Example 21, 67%.

Membranes formulated in accordance with Examples 19-22 were tested todetermine their permeability characteristics as a function of percentPVdC/AN by weight. Results are plotted in FIG. 6. Advantageous results,as noted above with respect to Examples 15-18, are realized.

EXAMPLES 23-46

A series of polyacrylate membranes were formulated. The percent byweight of each component present, based on the weight of the overallmembrane, is given for Examples 23-46 in Table 1.

A detailed description of the method of formulation of the compositionof Example 46 is given below.

9.46 g acrylonitrile having a molecular weight of 53.06, 19.71 g2-ethylhexylacrylate having a molecular weight of 184.28, 28.56 gmethylmethacrylate having a molecular weight of 100.00, 12.27 g vinylacetate having a molecular weight of 86.09 and 0.070 gazo-bis-isobutyronitrile as an initiator having a molecular weight of192.30 were dissolved to form a homogeneous solution. The startingmaterials were all either freshly distilled or recrystallized. Two glassplates were mounted so as to be parallel to each other, separated by aspace of 2 mm. The plates were sealed using a rubber gasket along threeedges, and the resultant form was filled with approximately 32 g of theabove solution. The glass plate form was then heated to 60° C. for 42hours in a nitrogen-flushed dry box. The mixture of monomers waspolymerized to a solid state. The solid polymer was then dissolved inabout 150 ml of chloroform, filtered through a glass filter, andprecipitated into four liters of methanol. The white precipitate wasthen dried in vacuo at 40° C. for three days.

The membranes of Examples 23-45 having the components in the proportionsgiven in Table 1 were formulated and obtained as described with respectto Example 46.

EXAMPLE 47

The following components, in the following amounts, were uniformlyadmixed and used in the fabrication of a gasket 66: A copolymer of anepichlorohydrin homopolymer and allyl glycidyl ether, available underthe trademark Grechon 1100 from Zeon Chemicals, 100 parts by weight;potassium stearate, 4 parts by weight; calcium oxide, 1 part by weight;stearic acid, 1 part by weight;2-ethyl-2-(hydroxymethyl)-1,3-propanediol trimethacrylate, sold ascatalog number SR-350 by Sartomer Co. of West Chester, Pa., 3 parts byweight; and 2,5-bis(t-butylperoxy)-2,5-dimethylhexane, sold under themark Varox DBPH-50, by R. T. Vanderbilt Co., Inc., 2 parts by weight.

A steel mold having a nickel-filled teflon coating available fromDav-Tech Plating, Inc., was employed. The gasket components were milledto form a 3/8 inch sheet and introduced into a mold having dimensions asdescribed above with respect to preferred sensor embodiments. Thecomponents were molded for 20 minutes at 170° C. under a mold force of9000 lbs. After molding, the mold was cooled via tap water and thegasket was removed.

EXAMPLE 48

A sensor 130 in accordance with Examples 1, 5, and 8 was fabricated,with the following exceptions. Conductive strip 34 was fabricated fromsilver paste available as part no. 6160 Ag from DuPont. Conductive strip38 was fabricated from a gold material available as part no. JM6990 fromJohnson-Matthey present in an amount of 95% by weight, and a glassbonding frit available as part number PC10129 from Metech, present in anamount of 5% by weight. After deposition of dielectric 40, the portionof conductive strip 34 defining reference electrode 50 waselectrochloridized to produce a silver/silver chloride reference. Atopthe electrolyte, lithium-exchanged Nafion was deposited on the sensorassembly to give a film thickness of about 0.010 mm. Thereover, membrane60 was deposited as an acrylonitrile-butadiene copolymer, available aspart no. 533 from Scientific Polymer Products. Subsequently, aconventional sample chamber was constructed above the sensor area usinga conventional gasket and a clear plastic cover. The sensor wasconnected to a potentiostat and the working and reference electrodeswere biased at a potential difference of approximately - 0.900 volts.Wetup to activate the Nafion electrolyte was effected by introducing asaline solution into the conventional sample chamber. Specifically, awash solution comprising 96.00 mM Nacl, 4.00 mM KCl, 1.25 mM Ca(OAc)₂,16.00 mM LiOAc, 44.00 mM NaOAc, a small amount of a microbicide, and asmall amount of a surfactant was introduced. Wetup rate was tested bycomparing the current flowing between the working and counter electrodesat 15 minutes following introduction of the saline solution and then at60 minutes following introduction. It was determined that theelectrolyte was actuated to 99% at the 15 minute point.

Subsequently, a series of aqueous oxygen calibrator solutions wereapplied to the sensor area by introducing them into the sample chamber.Specifically, calibrator solutions carrying oxygen contents of 0%, 12%,and 20% were successively introduced. Percent oxygen content values aregiven based on total gas dissolved in solution. Calibrator solutions arecommonly commercially available.

FIG. 7 shows the results of introduction of the aqueous oxygencalibrators, illustrated as a curve of current response versus time. Thesignificant result obtained using the sensor fabricated in accordancewith the example is the stable, linear current response at locations 72and 74 of FIG. 7, representing 12% and 20% oxygen samples, respectively.This stable, linear response demonstrates the non-depleting nature ofworking electrode 154, fabricated in accordance with the embodimentillustrated in FIGS. 2 and 4 using the laser hole or opening formationmethod. Thus, a combination of a small, laser-hole-formed workingelectrode with a cover membrane selected so as to advantageously passoxygen in limited quantities but pass water vapor very rapidly, with asolid electrolyte therebetween, results in a cumulative non-depletingsensor fabricated conveniently and economically. Inconsistencies 76 inFIG. 7 represent current anomalies resulting from serial manual sampleintroduction of a plurality of calibration samples.

EXAMPLE 49

Blood samples having predetermined pO₂ s were introduced into theconventional sample chamber of the sensor of Example 48, and the sensorwas polarized as in Example 47, following wetup and calibration.Specifically, tonometered blood samples carrying 12% oxygen and 20%oxygen, respectively, were introduced into the conventional samplechamber. FIG. 8 graphically illustrates current response versus time inthis experiment. Location 78 of the curve represents application of a12% aqueous oxygen calibrator solution, locations 80 represent currentresponse upon application of the 20% oxygen blood samples, and locations82 represent application of the 12% oxygen blood samples. Significantly,as noted above with respect to the calibration experiment illustrated inFIG. 7, the data illustrated in FIG. 8 shows the non-depleting nature ofthe sensor fabricated in accordance with one embodiment of the presentinvention, illustrated in that locations 80 and 82 show rapid, smooth,and repeatable equilibration of current response to oxygen content inthe blood. As in FIG. 7, in FIG. 8 anomalies 76 represent serialintroduction of samples into the sensor.

EXAMPLES 50-54

In these and following examples, the advantageous longevity of severalpreferred embodiments of the present invention will be demonstrated.Sensors were fabricated as follows: Examples 50 and 51, according toExamples 2, 3, 6, and 16; Example 52, according to Examples 2, 3, 6, and45; Examples 53 and 54, according to Examples 2, 3, 6, and 46.Subsequently, a sample chamber having a volume of approximately 1.5 μlwas constructed above the sensor area using a gasket in accordance withExample 47 and a cover member as described above. Each sensor wasattached to a potentiostat, set at a potential of -0.800 V between theworking and reference electrodes, and the sample chambers were filledwith the wash solution described above. Daily, each was flushed with0.1M aqueous NaCl and a linear sweep voltamogram (LSV) from 0 to -1.200V of each sensor was obtained, then each was flushed with the washsolution and again stored at -0.800 V potential. FIG. 9 graphicallyillustrates LSV's from 0 to 1.2 V obtained on day 16 of the experiment,plotted as current response as a function of applied potential. TheLSV's of Examples 52, 53, and 54 appear remarkably good in their smoothcurrent response that is substantially free of impurity and otherspurious peaks, while those of Examples 50 and 51 display significantspurious peaks. FIG. 10 illustrates response of these sensors to oxygencalibrator samples as described in Example 48, plotted as currentresponse as a function of PO₂. Included are plots 84, 86, and 88 of thelinearity of current response as a function of repeated calibratorapplication. The sensors of Examples 52, 53, and 54 show remarkablelinearity, and even the sensor of Example 51 shows relatively goodlinearity given its LSV illustrated in FIG. 9, demonstrating thatsatisfactory performance may be obtained despite moderate impurity peaksin voltamograms. FIG. 11 illustrates LSV's obtained on day 60 of thesensors of Examples 52, 53, and 54, showing extremely good stability ofthe sensors over time, evidenced by the lack of significant impurity andother spurious peaks.

EXAMPLES 55-57

In Examples 55-57, sensors were fabricated as follows: Example 55,according to Examples 2, 3, 6, and 42; Example 56, according to Examples2, 3, 6, and 45; Example 57, according to Examples 2, 3, 6, and 46.Subsequently, a sample chamber having a volume of approximately 1.5 ulwas constructed above the sensor area using a gasket in accordance withExample 47 and a cover member as described above. Each sensor wasattached to a potentiostat, set at a potential of -0.800 V between theworking and reference electrodes, and the sample chambers were filledwith the wash solution described above. Daily, each was flushed with afresh wash solution, and a 3/2 cyclic voltamogram (CV) between 0 and-1.200 V of each sensor was obtained. Each 3/2 cyclic voltamogram wascarried out by polarizing the sensor at 0 V vs. Ag/AgCl, sweeping thevoltage to -1.200 V, sweeping back to 0 V, and sweeping finally back to-1.200 V, monitoring and plotting current response as a function ofapplied voltage along the way. Then each was flushed with the washsolution and again stored at -0.800 V potential.

The results of these experiment on various days for Examples 55, 56, and57 are illustrated in FIGS. 12A-12G, 13A-13G, and 14A-14H, respectively.Excellent stability is demonstrated, as the 3/2 cyclic voltamograms donot change appreciably in appearance from one day's experiment toanother's. Indeed, all the 3/2 cyclic voltamograms show absence ofsubstantial impurity or other spurious peaks.

EXAMPLE 58

A sensor 30 was fabricated in accordance with Examples, 2, 3, and 6,with a membrane fabricated in accordance with Example 24 depositedthereover, with the following exceptions. Two layers of dielectric weredeposited, and the Nafion electrolyte was deposited in a thickness of 10μm. Subsequently, a sample chamber having a volume of approximately 1.5μl was constructed above the sensor using a gasket in accordance withExample 47 and a cover membrane as described above.

The sensor was subjected to five days of electrochemical testing, usingaqueous oxygen calibrator solutions, human whole blood, and human serum.Specifically, a feasibility study was carried out in which the sensorwas set at a predetermined potential, and data was collected on 30 wholeblood human samples at three different levels of oxygen for three daysin a row. The blood samples were collected and tonometered at 37° C. toone of three predetermined oxygen concentration levels. Data was notcollected from serum samples introduced into the sample chamber, butserum was introduced so as to subject the sensor to more human samples.Serum was run at ambient temperature and gas levels. Between each humanblood sample, human serum sample, and between each calibrator levelintroduction, the wash solution of Example 48 was introduced into thesample chamber. During the testing, the sensor was connected to apotentiostat and the working and reference electrodes were biased at apotential difference of approximately -0.800 volts. Data points werecollected every two seconds.

On day 1, wetup was effected by introducing the wash solution into thesample chamber. Then, a 32-minute calibration curve was obtained byintroducing a sample of a 5.99% oxygen calibrator into the samplechamber, followed by a 60-second data collection period, after which thestep was repeated. A 12.94% oxygen calibrator was then sampledidentically, followed by a 23.99% oxygen level calibrator, sampledidentically.

On each of days 2-4, the following protocol was carried out. First acalibration step as described above with regard to the day 1 dataacquisition was carried out. Then, a 50-minute protocol was carried outby introducing the wash solution into the sample chamber, followed by atwo-minute data collection period, followed by introduction of humanserum at ambient oxygen concentration, followed by a two-minute datacollection period. The wash/serum sequence was repeated ten times,ending with a wash step. The entire serum protocol was repeated for atotal of 20 serum samples. Following the serum protocol, theabove-described calibration protocol was run. Then a 47-minute humanwhole blood protocol was run. Specifically, a 5.99% oxygen calibratorwas introduced into the sample chamber, followed by a one-minute datacollection period. This sequence was repeated with a 12.94% oxygencalibrator, and then with a 23.99% oxygen calibrator. Subsequently, thewash solution was introduced into the sample chamber and a 90-seconddata collection was carried out. Then a sample of whole bloodtonometered to 21% oxygen was introduced into the sample chamber,followed by a 60-second data collection period, followed by introductionof the wash solution, followed by introduction of the tonometered wholeblood sample. The sequence was repeated until 10 whole blood samples hadbeen introduced. The above-noted calibration protocol was then run. Thenthe whole blood protocol was repeated, first with a 12.94% oxygentonometered whole blood sample, and then with a 7% oxygen tonometeredwhole blood sample. At the end of this protocol, the calibrationprotocol was repeated.

On day 5, the above-noted calibration protocol was repeated. Then, thesensor was evaluated by obtaining a sensor polarogram as described abovewith respect to Examples 55-57, with the exception that 1/2 of a cyclicvoltamogram was run. The polarogram was run from 0 to -1.200 volts.

FIG. 15 illustrates results of the human whole blood tonometered samplesexposed to the sensor as described above. The data was collected on day2 of the test. Introduction of the 5.99% oxygen calibrator isrepresented at 90, introduction of the 12.94% oxygen calibrator isrepresented at 92, and introduction of the 23.99% oxygen calibrator isrepresented at 94. Introduction of the wash solution is represented at94. Introduction of the wash solution is represented at locations 96.Introduction of the whole blood samples tonometered to 12.94% oxygen arerepresented by locations 98. The discrepancy between locations 98,representing 12.94% oxygen tonometered whole blood, and locations 92,representing 12.94% oxygen calibrator, is due to the well-knowncharacteristic of blood in producing lower oxygen readings thancalibrator solutions. Adjustment for such discrepancy is easily made bythose of ordinary skill in the art. Significant results illustrated inFIG. 15 include the good precision in current response to oxygen levels,and good linearity in the calibration step observed.

FIG. 16 illustrates three representative polarograms obtained on day 5of the test, illustrating good durability of the sensor.

The preceding examples are set forth to illustrate specific embodimentsof the invention and are not intended to limit the scope of theinvention. Additional embodiments and advantages within the scope of theclaimed invention, for example measuring electroactive species otherthan oxygen with sensing means described herein, and employingcomponents of the inventive sensor for purposes other thanelectrochemical analysis, will be apparent to those of ordinary skill inthe art.

                  TABLE 1    ______________________________________    Ex.    No.  1       2      3    4    5    6    7    8    9    ______________________________________    23   72.8    50%    10%  40%    24   45.1    60%    20%  20%    25   39.5    70%    30%    26   21.5    60%    40%    27   11.6    30%    50%  20%    28   17.4    40%         20%  40%    29   -3.9    40%    40%            20%    30   -17.9   30%    30%            40%    31   -15.6   30%              40%  30%    32   18.3    60%              30%  10%    33   54.8                70%                 30%    34   84.9           20%  80%    35   47.3           40%  40%            20%    36   30.1    50%    40%                           10%    37   28.1    40%    50%                           10%    38   15.4    30%    60%                           10%    39   43.0    52%    35%                           13%    40   38.7    44%    40%                           16%    41   29      55%    10%  10%  15%  10%    42   18      40%    20%  20%  15%   5%    43   32      40%    20%  30%       10%    44   34      30%    25%  40%        5%    45   59      25%    10%  40%       25%    46   42      25%    15%  40%       20%    ______________________________________     1  Tg (°C.)     2  Acrylonitrile     3  2ethylhexylmethacrylate     4  methylmethacrylate     5  dodecylmethacrylate     6  vinylacetate     7  cyclohexylmethacrylate     8  2hydroxypropylmethacrylate     9  acrylamide

What is claimed is:
 1. A method for selecting an electrically-conductivematerial for use in an electrochemical sensor, comprising:providing asensor for electrochemical analysis, said sensor including anelectrically-conductive material, measuring the polarogram of saidmaterial to determine the shape of said polarogram in a set potentialregion within which said sensor is designed to operate, and evaluatingsaid polarogram in said set potential region to determine whether saidpolarogram exhibits a plateau, whereby the utility of said material isdetermined.
 2. A method for evaluating the durability of anelectrochemical sensor comprising:providing a sensor for electrochemicalanalysis, measuring the polarogram of said sensor to determine the shapeof said polarogram in a set potential region within which said sensor isdesigned to operate, and evaluating said polarogram in said setpotential region to determine whether said polarogram exhibits asubstantially flat current vs. voltage plot, whereby the durability ofthe sensor is determined.
 3. A method for selecting anelectrically-conductive material for use in an analytical sensor,comprising:providing a sensor for electrochemical analysis, said sensorincluding an electrically-conductive material, measuring the polarogramof said material to determine the shape of said polarogram in a setpotential region within which said sensor is designed to operate, andevaluating said polarogram in said set potential region to determinewhether said polarogram exhibits a substantially flat current vs.voltage plot, whereby the utility of said material is determined.
 4. Amethod for evaluating the durability of an electrochemical sensor,comprising:providing a sensor for electrochemical analysis, measuringthe polarogram of said sensor to determine the shape of said polarogramin a set potential region within which said sensor operates, andevaluating said polarogram in said set potential region to determinewhether the voltage fluctuation varies outside a margin of error toassess the durability of said sensor.